Neutralizing GP41 antibodies and their use

ABSTRACT

Monoclonal neutralizing antibodies are disclosed that specifically bind to the HIV-1 gp41 membrane-proximal external region (MPER). Also disclosed are compositions including the disclosed antibodies that specifically bind gp41, nucleic acids encoding these antibodies, expression vectors including the nucleic acids, and isolated host cells that express the nucleic acids. The antibodies and compositions disclosed herein can be used for detecting the presence of HIV-1 in a biological sample, or detecting an HIV-1 infection or diagnosing AIDS in a subject. In additional, the broad neutralization breadth of the disclosed antibodies makes them ideal for treating a subject with an HIV infection. Thus, disclosed are methods of treating and/or preventing HIV infection.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No.PCT/US2012/063958, filed Nov. 7, 2012, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 61/702,703, filed Sep. 18, 2012; U.S.Provisional Application No. 61/698,480, filed Sep. 7, 2012; U.S.Provisional Application No. 61/672,708, filed Jul. 17, 2012; and U.S.Provisional Application No. 61/556,660, filed Nov. 7, 2011. Each ofthese prior applications is incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

This relates to the identification of monoclonal neutralizingantibodies, such as, but not limited to, antibodies that bind to themembrane-proximal region of HIV-1 gp41.

BACKGROUND

An effective Human Immunodeficiency Virus type 1 (HIV-1) vaccine willlikely need to induce neutralizing antibodies (NAbs) that block HIV-1entry into human cells. To be effective, vaccine-induced antibodies willhave to be active against most circulating strains of HIV-1.Unfortunately, current HIV-1 vaccines are unable to induce potent andbroadly reactive NAbs. One major obstacle to the design of bettervaccines is the limited understanding of what region of the HIV-1envelope glycoproteins, such as gp120 and gp41, are recognized by NAbs.A few neutralizing monoclonal antibodies (mAbs) have been isolated fromHIV-1 infected individuals and these mAbs define specific regions(epitopes) on the virus that are vulnerable to NAbs.

Although the envelope glycoproteins are immunogenic and induce a varietyof antibodies, the neutralizing antibodies that are induced arestrain-specific, and the majority of the immune response is diverted tonon-neutralizing determinants (Weiss, R. A., et al., Nature, 1985. 316(6023): p. 69-72; Wyatt, R. and J. Sodroski, Science, 1998. 280 (5371):p. 1884-8). Broadly neutralizing antibodies have been isolated onlyrarely from natural HIV infection. Three examples of broadlyneutralizing antibodies that bind gp41 are 2F5, 4E10 and Z13E1. Thesegp41 neutralizing antibodies recognize the membrane-proximal region(MPER) of the HIV-1 gp41 glycoprotein. Unfortunately, these antibodiesare limited in their strain cross reactivity or their potency andtherefore do not provide a viable choice for therapeutic intervention.Thus, the need exists for methods to prepare monoclonal broadlyneutralizing antibodies that can provide protection from an infectiousagent, such as HIV.

SUMMARY

Isolated human monoclonal neutralizing antibodies that specifically bindgp41 are provided herein. In certain examples, the binding and/orneutralization ability of these antibodies has been optimized. Alsodisclosed herein are compositions including the disclosed antibodiesthat specifically bind gp41, nucleic acids encoding these antibodies,expression vectors including the nucleic acids, and isolated host cellsthat express the nucleic acids. Antigen binding fragments of theisolated antibodies are also provided.

In some embodiments, an isolated human monoclonal antibody, or antigenbinding fragment thereof, includes a heavy chain and a light chain,wherein the heavy chain includes an amino acid sequence at least about80% identical to the amino acid sequence set forth as SEQ ID NO: 1. Inseveral such embodiments, the antibody, or antigen binding fragmentthereof, specifically binds to gp41 and contacts L, WF, LW and R in theamino acid sequence set forth as LWNWFDITNWLWYIR (SEQ ID NO: 26,residues 14-28), and is neutralizing. In additional embodiments, theantibody, or antigen binding fragment thereof, specifically binds togp41 and contacts NWF, T, and R in the amino acid sequence set forth asNWFDITNWLWYIR (SEQ ID NO: 13, residues 7-19), and is neutralizing. Inadditional embodiments, an isolated monoclonal antibody or antigenbinding fragment is provided that includes a heavy chain and a lightchain, wherein the heavy chain includes amino acids 26-33 (heavy chaincomplementarity-determining region 1 (HCDR1)), 51-60 (HCDR2), or 99-120(HCDR3) of SEQ ID NO: 11, wherein X₁ is Q or R, X₂ is V or A, X₃ is S orY, and X₄ is T or I. The antibody or the antigen binding fragmentspecifically binds gp41 of HIV-1, and is neutralizing. In some suchembodiments, the isolated human monoclonal antibody or antigen bindingfragment includes a heavy chain including one or more of amino acids26-33 (HCDR1), 51-60 (HCDR2), and 99-120 (HCDR3) of one of SEQ ID NOs:1, 3, 5, 147-149, 189-192, or 200-204. In some such embodiments, theheavy chain of the isolated human monoclonal antibody includes an aminoacid sequence at least 90% identical to the amino acid sequence setforth as one of SEQ ID NOs: 1, 3, 5, 147-149, 189-192, or 200-204. Inother embodiments, the heavy chain of the isolated human monoclonalantibody or antigen binding fragment thereof, includes the amino acidsequence set forth as one of SEQ ID NOs: 1, 3, 5, 147-149, 189-192, or200-204.

In additional embodiments, the isolated human monoclonal antibody orantigen binding fragment thereof includes a light chain at least about80% identical to the amino acid sequence- set forth as SEQ ID NO: 2. Infurther embodiments, the light chain includes amino acids 26-31 (lightchain complementarity-determining region 1 (LCDR1)), 49-51 (LCDR2), and88-99 (LCDR3) of SEQ ID NO: 12, where X₄ is E or D, X₅ is Y or H, X₆ isK or I, X₇ is V or I, X₈ is S or T, X₉ is D or E, X₁₀ is E or D, and X₁₁is T or I. In additional embodiments, the light chain includes aminoacids 26-31 (LCDR1), 49-51 (LCDR2), or 88-99 (LCDR3) of one of SEQ IDNO: 2, 4, 6, 12, 150-152, or 164-186. In some embodiments, the lightchain of the isolated human monoclonal antibody or antigen bindingfragment includes an amino acid sequence at least 90% identical to theamino acid sequence set forth as one of SEQ ID NO: 2, 4, 6, 12, 150-152,or 164-186. In one embodiment, the light chain of the isolated humanmonoclonal antibody or antigen binding fragment includes the amino acidsequence set forth as one of SEQ ID NO: 2, 4, 6, 12, 150-152, or164-186.

In some embodiments, an isolated human monoclonal antibody or antigenbinding fragment thereof is provided in which the heavy chain includesthe amino acid sequence set forth as SEQ ID NO: 1 and the light chainincludes the amino acid sequence set forth as SEQ ID NO: 2. The antibodyspecifically binds gp41 of HIV-1 and is neutralizing. In furtherembodiments, an isolated human monoclonal antibody or antigen bindingfragment thereof is provided in which the heavy chain includes the aminoacid sequence set forth as SEQ ID NO: 154 and the light chain includesthe amino acid sequence set forth as SEQ ID NO: 152. The antibodyspecifically binds gp41 of HIV-1 and is neutralizing. In otherembodiments, an isolated human monoclonal antibody or antigen bindingfragment thereof is provided in which the heavy chain includes the aminoacid sequence set forth as SEQ ID NO: 192 and the light chain includesthe amino acid sequence set forth as SEQ ID NO: 152. The antibodyspecifically binds gp41 of HIV-1 and is neutralizing.

The antibodies and compositions disclosed herein can be used for avariety of purposes, such as for detecting the presence of HIV-1 in abiological sample or diagnosing AIDS. These methods can includecontacting a sample from a subject with a human monoclonal antibody thatspecifically binds gp41, and detecting binding of the antibody to thesample. An increase in binding of the antibody to the sample relative tobinding of the antibody to a control sample identifies the subject as asubject with HIV-1 infection and/or AIDS. In some non-limiting examples,an increase in binding of the antibody to the sample relative to acontrol sample detects the presence of HIV-1.

Method are also disclosed for treating a subject with an HIV infection,such as, but not limited to, a subject with AIDS. The methods includeadministering a therapeutically effective amount of a monoclonalantibody as described above to a subject.

The foregoing and other features and advantages of this disclosure willbecome more apparent from the following detailed description of severalembodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C are as set of tables and a diagram illustrating analyses of10E8 antibody sequence and neutralization. (A) Inferred germline genesencoding the variable regions of 10E8, 7H6 and 7N16. (B) Neutralizingactivity of antibodies against a 181-isolate HIV-1 envelope protein(Env)-pseudovirus panel. Dendrograms indicate the gp160 protein distanceof HIV-1 primary isolate Envs. (C) Data below the dendrogram show thenumber of tested viruses, the percentage of viruses neutralized, and thegeometric mean IC50 for viruses neutralized with an IC50<50 μg/ml.Median titers are based on all tested viruses, including those withIC50 >50 ug/ml, which were assigned a value of 100.

FIGS. 2A and 2B illustrate the binding specificity of 10E8. (A)Enzyme-Linked immunosorbant assay (ELISA) binding of mAb 10E8 or 4E10 togp140, gp120, gp41, or 4E10 peptide. Error bars denote one standarderror of the mean (SEM). (B) Inhibition of mAbs 10E8 or 4E10neutralization of C1 HIV-2/HIV-1 MPER virus by 4E10 alanine scanningpeptides. Peptide was incubated with mAb 4E10 or 10E8 for one hour priorto infecting TZM-b1 cells. Y-axis shows percent neutralization for eachcondition. W₆₇₂, F₆₇₃, T₆₇₆ and R₆₈₃ residues were positions for whichthe alanine mutant peptide did not block neutralization (R₆₈₃ only forthe 10E8 antibody). Residues 16-28 of SEQ ID NO: 26 are shown.

FIGS. 3A and 3B are a set of graphs and a set of digital imagesillustrating analysis of 10E8 autoreactivity. (A) Surface PlasmonResonance (SPR) analysis of 10E8 binding to anionic phospholipids. 10E8was injected over PC-CLP liposomes or PC-PS liposome immobilized on theBIACORE® L1 sensor chip. 4E10 and 2F5 were used as positive controls and13H1, 17b, and anti-RSV F protein as negative controls. (B) Reactivityof 10E8 with HEP-2 epithelial cells. Controls are as above with VRC01added as an additional negative control. Antibody concentration was 25μg/ml. All pictures are shown at 400× magnification.

FIGS. 4A-4H are a set of ribbon diagrams illustrating the crystalstructure of 10E8 antibody in complex with its gp41MPER epitope. (A)10E8 recognizes a highly conserved gp41 helix to neutralize HIV-1. Fab10E8 is shown in ribbon representation (shades of dark grey for heavychain and of light grey for light chain) in complex with a gp41 peptide(dark grey) that encompasses the MPER (Asn₆₅₆-Arg₆₈₃;NEQELLELDKWASLWNWFDITNWLWYIR (SEQ ID NO: 26)). (B) Interface between10E8 and gp41 with select 10E8-side chains and gp41-side chains in stickrepresentation. In analogy to a hand, the hinge can be viewed as beinggripped by a thumb (represented by the CDR H2), the C-terminal helix asbeing suspended along a corresponding extended forefinger (representedby the CDR H3), and residues that commence the C-terminal helix as beingcaught in the cleft between the thumb and forefinger (represented by thejuncture of the CDR loops). (C-D) Buried contact surfaces and epitopeconservation. An examination of the buried contact surface on gp41(grey; C) reveals that epitope residues (labeled, D) that are directlycontacted by 10E8 are highly conserved across 2870 examined strains(conservation percentages provided in parentheses; see also FIGS.26-28). E-H, Alanine mutagenesis of paratope and epitope. Residues atthe tip of the 10E8 CDR H3 loop and within the hydrophobic cleft arecrucial for recognition of gp41 and for virus neutralization (FIGS.31-32), as mapped onto the buried 10E8-contact surface (E, G). Theseresults mirror the effects of alanine scan mutations of the 10E8 epitope(FIGS. 18-19), as mapped onto the buried gp41-contact surface (F, H). Acomparison of the effects of the alanine mutagenesis of the paratope andepitope reveal that residues of the epitope that are most crucial for10E8 recognition and neutralization, are also the most highly conserved(D).

FIGS. 5A and 5B are a table, a set of graphs and a schematic diagramillustrating a site of gp41 vulnerability. (A) Impact of sequencevariation on 10E8 neutralization. Predicted amino acid sequences withinthe binding epitope of 10E8 for three 10E8 resistant viruses and thepatient virus are shown. The 10E8 binding region and differences insequence compared to the JR2 virus are labeled in light grey. IC50 andIC80 values that are >20-fold than JR2 wild-type pseudovirus arehighlighted in light grey. Error bars denote one SEM. (B) Structuraldefinition of a highly conserved region of gp41 recognized byneutralizing antibodies. Atoms of highly conserved residues that makedirect contacts with 10E8 are shaded medium grey and shown in stickrepresentation, atoms buried by 10E8 are shaded dark grey, and mainchain-contacting atoms are shaded light grey. Semi-transparent surfacesof the gp41MPER are shaded according to the underlying atoms. 90° viewsare shown, with bound antibody 10E8 in the right panel. The 10E8 CDR H3interacts with highly conserved hydrophobic residues, whereas the CDR H2contacts main chain atoms at the juncture between the N- and C-terminalhelices. Many of the unbound residues of the MPER (grey) arehydrophobic, especially those within the C-terminal helix. In thestructure of a late fusion intermediate (FIG. 16) these residues facetowards the outside of a helical coiled-coil; in the pre-fusionconformation of the viral spike, these may interact with the viralmembrane or with other hydrophobic regions of Env.

FIGS. 6A and 6B depict a sequence alignment of the heavy and lightchains of gp41 antibodies 10E8 (SEQ ID NO: 1 and 2), 7H6 (SEQ ID NO: 3and 4), 7N16 (SEQ ID NO: 5 and 6), IGHV3-15*05 (SEQ ID NO: 7) and thegermline sequence of IGLV3-19*01 (SEQ ID NO: 8). Residues in light greyrepresent substitutions from the germline sequence. The dot symboldenotes the residue deletion. Kabat and IMGT numbering are shown and areused to identify specific residues in the 10E8 heavy and light chains.

FIG. 7 is a graph illustrating the correlation of neutralizationpotencies between N152 donor serum and antibody 10E8. Shown is a plot ofthe neutralization ID50s of N152 donor serum against neutralizationIC50s of 10E8, assessed against a 20-pseudovirus panel. A non-parametricSpearman correlation was used to evaluate the correlation between IC50sof 10E8 and ID50s of N152.

FIGS. 8A-8C are a graph and a set of tables illustrating the bindingspecificity of 10E8. (A) ELISA binding of indicated mAbs to MPER, 2F5,Z13e1, 4E10 and 4E10.19 peptides. Amino acid sequences of peptides arealso shown (descending, SEQ ID NO: 26 with N- and C-terminal lysinetriplicates, residues 1-16 of SEQ ID NO: 26, residues 11-21 of SEQ IDNO: 26 with a C-terminal lysine triplicate, residues 16-24 of SEQ ID NO:26 with a C-terminal lysine triplicate and residues 16-28 of SEQ ID NO:26 with a N-terminal cysteine and a C-terminal lysine triplicate). (B-C)Inhibition of mAb neutralization of C1 HIV-2/HIV-1 MPER chimeric virusby addition of MPER, 2F5, Z13e1, 4E10 and 4E10.19 peptides. Fold effectwas calculated as the ratio of neutralization IC50 with mockpeptide/IC50 (B), or the ratio of neutralization IC80 with mockpeptide/IC80 (C), for the indicated peptide. Values >5 are shaded lightgrey.

FIGS. 9A and 9B illustrate surface-plasmon resonance analysis of bindingof 10E8, 2F5 and 4E10 antibodies to a gp41MPER peptide. (A) Abiotinylated MPER peptide comprised of residues 656-683 of gp41 wasimmobilized on a streptavidin SA chip (GE Healthcare) and antibody Fabsflowed over as analyte at 2-fold serial increasing concentrationsranging from 3.9-125 nM (10E8), 0.49-31.25 nM (2F5), and 0.25 nM to 62.5nM (4E10). Association and dissociation phases of three minutes and fiveminutes, respectively, were used, at a flow rate of 30 μl/minute, andeach analyte concentration was performed in triplicate. (B) Bindingconstants, listed, were obtained by fitting sensograms with a 1:1Langmuir model. The amino acid sequence of SEQ ID NO: 26 is shown.

FIG. 10 is a pie chart illustrating the frequency of HIV-1+sera with agiven specificity. Sera from 78 healthy HIV-1-infected donors were usedin this assay. Frequency measured by the ability of patient sera toneutralize HIV-2/HIV-1 chimeras containing portions of the MPER andconfirmed with peptide blocking. Neutralization ID50s of sera arereported in FIG. 21. Fold changes of ID50 after peptide blocking arereported in FIG. 22. The six patients with sera containing 10E8-likeantibodies were not different from the remaining 72 patients with regardto viral load (6748 copies per ml with 10E8 vs 5446 without; p>0.05),CD4 count (437 cells/μl vs. 557; p>0.05), years since diagnosis (20years vs. 13; p>0.05), or median neutralization titer (302 vs. 156;p>0.05).

FIGS. 11A-11C illustrate accessibility of 10E8 to the MPER. (A) Bindingof 10E8, 4E10 and 2F5 to full-length HIV_(JR-FL) envelope spikes, 4E10mutant (Phe673Ser) or 2F5 mutant (Lys665Glu) expressed on 293T cellsurface as measured by flow cytometry. Serially diluted antibody wasincubated with cells for one hour. 2G12 and b12 antibodies were used aspositive controls and F105 was used as a negative control. VRC01 is usedas an additional control on JR-FL transfected cells. Relative bindingpercent is calculated as the mean fluorescence intensity (MFI) dividedby the maximum MFI of the positive control 2G12×100. (B) MPERaccessibility was determined by washing antibody-virion mixture prior toinfecting TZM-b1 cells. Pseudoviruses were incubated with antibodies at37° C. for 30 minutes, and antibody-virion mixture was washed or notwashed prior to infecting target cells. (C) Impact of washing onantibody neutralization was measured by the area under the curve (AUC)or at the IC80. For BaL and JRF1 the IC80 was not achieved in theno-wash condition and the highest inhibitory concentration (IC60 andIC75, respectively) was used.

FIGS. 12A and 12B are schematic diagrams illustrating comparison of twocopies of the gp41 MPER in the crystal asymmetric unit. (A) gp41peptides from the two 10E8-gp41 complexes in the crystal asymmetric unitare shown in stick representation (complex 1, dark grey; complex 2,medium grey), surrounded by their 2fo-fc electron density contoured at16 (dark grey). Images shown are rotated 180° relative to each other,and are in the same orientation as in FIGS. 4C and 4D. (B) An alignmentof the peptides in the two crystalline complexes. Shown, at 90° views,is a superposition of the two peptides in the asymmetric unit based onalignment of all atoms of residues 671-683. The N-terminal helix incomplex 2 is shifted by 45° relative to the one in complex 1 in thisalignment. While the differing orientations of the N-terminal helix inthe two complexes suggests a degree of structural plasticity, residuesof the hinge and C-terminal helix in the two complexes are highlyconserved and are involved in the most critical interactions with theantibody.

FIGS. 13A-13C are a set of graphs illustrating surface-Plasmon resonanceanalysis of 10E8 paratope alanine variants. Shown are binding sensogromsof MPER peptide to 25 10E8 paratope variants, as well as to wild-type(wt) 10E8. Variant IgGs were captured on an anti-human IgG-coupledbiosensor chip to surface densities of 1000-2000 response units and MPERpeptide (listed) flowed over analyte at serial twofold dilutionsstarting at 500 nM (with the exception of HC D30A, W100bA, S100cA,P100fA, which started at 250 nM). Association and dissociation phases ofthree minutes and five minutes, respectively, were used, at flow ratesof 30 μl/min. Sensograms were fit with a 1:1 Langmuir model usingBIACORE® BIAEVALUATION® Software (GE Healthcare). Binding constants arereported in FIG. 31. The amino acid sequence of SEQ ID NO: 26 is shownin FIG. 13C.

FIG. 14 shows a graph illustrating the correlation of 10E8 variantbinding and neutralization. Binding K_(D)'s of the 10E8 alanine paratopevariants plotted against their mean neutralization IC50s and IC80s. Anonparametric Spearman correlation was used to assess the relationshipbetween binding and neutralization. K_(D)'s and neutralization IC50s andIC80s are reported in FIGS. 31-32.

FIGS. 15A-15H illustrate recognition of a structurally conservedC-terminal MPER helix by 10E8 and 4E10. 10E8 and 4E10 use substantiallydifferent modes of recognition to bind a structurally conserved helix atthe C-terminus of the gp41MPER. (A) MPER conformation and buried surfacefor neutralizing antibodies 10E8, 2F5 (Protein DataBank (PDB) ID No.1TJI, incorporated by reference herein as present in the database onOct. 22, 2012), Z13e1 (PDB ID No. 3FN0, incorporated by reference hereinas present in the database on Oct. 22, 2012), and 4E10 (PDB ID No. 2FX7,incorporated by reference herein as present in the database on Oct. 22,2012). Cα-ribbon representations of the gp41MPER bound by each antibodyare shown, with bar graphs displaying the amount of buried surface perresidue. The amino acid sequence of SEQ ID NO: 26 is shown below eachbar graph. The sequence of the crystallized epitope is shown inuppercase letters. (B) Superposition of the MPER C-terminal helix, inthe 10E8-(dark grey) and 4E10-bound (light grey) conformations,displayed at 90° orientations. Cα-ribbon representations are shown, withside chains displayed as sticks. (C-F) Comparison of 10E8 and 4E10recognition of the C-terminal MPER helix, with molecules displayed inCα-ribbon representations. (C) 10E8 variable domains in complex with theMPER, shaded according to FIG. 4A. (D) 4E10 variable domains in complexwith the MPER (PDB ID No. 2FX7, incorporated by reference herein aspresent in the database on Oct. 22, 2012), in an orientation based onthe alignment of gp41 C-terminal helices described in (B; left panel).gp41 is shaded off-white, 4E10 heavy chain, dark grey, and 4E10 lightchain, medium grey. (E-F) 90° views of (C) and (D), looking down fromC-terminus to N-terminus of the conserved MPER C-terminal helix. (G,H)Helical wheel representations of the 10E8- and 4E10-bound MPERC-terminal helices, reflecting the orientation displayed in (B; rightpanel). Highlighted antibody-contacting faces are based on directcontacts observed between the antibodies and gp41, as described in FIG.35.

FIG. 16 shows a ribbon diagram illustrating the gp41 site ofvulnerability mapped onto a “late intermediate” conformation of gp41.The gp41 site of vulnerability as defined by the contact footprint ofantibody 10E8 on gp41 is shown mapped onto the 10E8-bound MPER peptidestructure (left, similar orientation as FIG. 6B) and a late intermediatesix-helix bundle conformation of gp41 (PDB ID No. 2XR7 (right),incorporated by reference herein as present in the database on Oct. 22,2012). Atoms recognized by antibody 10E8 are shaded dark grey and shownin stick representation. Atom or residue contacts exclusive toantibodies 2F5, Z13e1, and 4E10, are shown as sticks, shaded light ormedium grey. The 10E8-defined site of vulnerability faces outwards, awayfrom the core axis of the bundle, and appears to be largely accessiblein this conformation. A potential N-linked glycosylation site atposition 674 would not be compatible with the late intermediateconformation since the sidechain of residue 674 faces the interior ofthe six-helix bundle.

FIGS. 17A-17F are a set of tables illustrating 10E8 neutralizingproperties. (A) Neutralization of 10E8 and 7H6 against a 5-isolateEnv-pseudovirus mini-panel. IC50 values of less than 1 μg/ml arehighlighted in grey. (B) Neutralization profile of patient N152 serumand monoclonal antibodies. ^(a)The data for N152 shows the ID50 (dose ofvirus required for 50% infection) of serum against each virus. ID50>1000is highlighted in dark grey, 500<ID50<1000 is in medium grey, and100<ID50<500 is in light grey. The data for the monoclonal antibodiesshows the IC50. IC50 <1 μg/ml is highlighted in medium grey; IC50 of1-10 μg/ml is highlighted in light grey; and IC50 of 10-50 μg/ml ishighlighted in dark grey. (C-F) Antibody neutralization data against 181HIV-1 Env-pseudoviruses. IC50 <1 μg/ml is highlighted in medium grey;IC50 of 1-10 μg/ml is highlighted in light grey; and IC50 of 10-50 μg/mlis highlighted in dark grey.

FIG. 18 is a table illustrating binding of 10E8 and 4E10 to gp41MPERalanine scanned peptides, by ELISA. Fold change was calculated aspeptide IC50/mock peptide IC50. Fold change values >10 are highlightedin light grey.

FIG. 19 is a table listing neutralization data for 10E8 againstpseudotyped HIV-1_(JR2) MPER alanine mutants. Concentration is μg/ml.IC50 and IC90 values >20-fold compared to JR2 wild-type for 10E8 or >100fold for 4E10 are highlighted in light grey.

FIG. 20 is a table listing sequences of the HIV-2/HIV-1 chimeras usedfor neutralization assays. The sequences of 7312A, C1, C1C, C3, C7, C6,C4, C4GW and C8 are shown (SEQ ID NOs: 15-22, respectively). Fragmentsof the MPER sequence that correspond to the sequence of the HIV-1 MPERare underlined.

FIG. 21 is a table illustrating mapping anti-MPER neutralizingantibodies/sera with HIV-2/HIV-1 chimeras. ^(a)IC50 (μg/ml) is shown.^(b)ID50 values are shown. Numbers are bolded and highlighted light greyif the ID50 of the HIV-2/HIV-1 chimera was both 3-fold greater thanHIV-2 wild-type control and >100. “-” indicates no neutralization. “ND”indicates the serum classification could not be determined.

FIG. 22 is a table illustrating blocking mAb- and serum-mediatedneutralization of the HIV-2/HIV-1 chimera C1 using mutant MPER peptides.The sequence of the blocking peptides is shown in FIG. 8A. ^(b)Foldchange of IC50 refers to (peptide IC50)/(mock peptide IC50). ^(c)Foldchange of ID50 refers to (Mock peptide ID50)/(peptide ID50). Light greyhighlight indicates a 3-fold change in IC50/ID50 relative to the controlpeptide.

FIG. 23 is a table illustrating reactivity of 10E8 with autoantigens.Reactivity of 10E8 with autoantigens was detected by the Luminex assay.An anti-RSV monoclonal antibody, Synagis (MedImmune, Gaithersburg, Md.),was used as a negative control. The 4E10, 2F5, VRC01 and 17b antibodieswere also tested for comparison. SSA refers to Sjogren's syndromeantigen A; SSB refers to Sjogren syndrome antigen B; Sm refers to Smithantigen; RNP refers to ribonucleoprotein; Sc1 70 refers to scleroderma70; Jo1 refers to antigen; CentrB refers to centromere B.

FIG. 24 is a table listing data collection and refinement statistics forthe 10E8 crystal structure studies. The highest resolution shell isshown in parentheses. The dataset shown was collected from one crystal.

FIG. 25 is a table listing Phi-Psi angles of antibody-bound gp41peptides. ^(a)For the 4E10:gp41 complex the structure of PDB ID No. 2FX7was used (incorporated by reference herein as present in the database onOct. 22, 2012). ^(b)For the Z13e1:gp41 complex the structure of PDB IDNo. 3FN0 was used (incorporated by reference herein as present in thedatabase on Oct. 22, 2012).

FIG. 26 is a table listing the total buried surface area on 10E8 andgp41. All interactions were performed using PISA(ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver). ^(§)BSA refers to BuriedSurface Area, Å².

FIG. 27 is a table listing buried surface areas at interface between10E8 heavy chain and gp41, by residue. All interactions were performedusing PISA (ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver). ^(£)Percentidentity of this residue in an analysis of 2870 HIV strains deposited inthe Los Alamos HIV sequence database (as of December 2011). ^(‡)ASArefers to Accessible Surface Area, Å². ^(§)BSA refers to Buried SurfaceArea, Å². Bars represent buried area percentage, one bar per 10%. Δ^(i)Grefers to Solvation energy effect, kcal/mol.

FIG. 28 is a table listing buried surface areas at interface between10E8 light chain and gp41, by residue. All interactions were performedwith PISA (ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver). ^(£)Percentidentity of this residue in an analysis of 2870 HIV strains deposited inthe Los Alamos HIV sequence database (as of December 2011). ASA refersto Accessible Surface Area, Å². BSA refers to Buried Surface Area, Å².Bars represent buried area percentage, one bar per 10%. Δ^(i)G refers toSolvation energy effect, kcal/mol.

FIG. 29 is a table listing hydrogen bonds and salt bridges between 10E8and gp41. Hydrogen bonds were determined with the use of the programLigplot (McDonald et al., J Mol Biol 238, 777-793, 1994). Chain H refersto heavy chain complex 1; Chain B refers to heavy chain complex 2; ChainP refers to gp41 peptide complex 1; Chain F refers to gp41 peptidecomplex 2.

FIG. 30 is a table listing Van der Waals contacts between 10E8 and gp41.Van der Waals contacts were determined with the use of the programLigplot (McDonald et al., J Mol Biol 238, 777-793, 1994). Chain H andchain L refer to complex 1 heavy and light chains, respectively; Chain Prefers to complex 1 gp41 peptide. Chain B and chain D refer to complex 2heavy and light chains, respectively; Chain F refers to complex 2 gp41peptide.

FIG. 31 is a table illustrating binding affinities of 10E8 alaninevariants to a soluble MPER peptide. SE refers to standard error; nbrefers to weak to undetectable binding at concentration range used.^Fold is defined as fold-change relative to individual wild type 10E8runs performed in parallel with variants. ^($)Mean of three individualwild type 10E8 runs. ^(#)Only those mutations that yielded effectsgreater than 10-fold were mapped onto the 10E8 buried surface in FIG. 4E(medium grey, >100×; light grey, 50×<100×; dark grey, 10×<50×). Heavychain residues Y99A_(HC) and G100hA_(HC) showed almost no binding tosoluble peptide, while mutations of additional residues of the CDR H3(F100aA_(HC), G100dA_(HC), P100fA_(HC), P100gA_(HC), E100iA_(HC), andE100jA_(HC)) decreased affinity 50-120 fold (Kabat numbering is used toidentify specific residues in the 10E8 heavy and light chains). CDR H1loop and framework region 2 mutations W33A_(HC) and R50A_(HC),respectively, which are present within the hydrophobic cleft, alsoknocked out binding to the MPER peptide, and the CDR H2 mutationE53A_(HC) within the cleft decreased affinity for the MPER peptide by60-fold. Light chain residue R91LC, which is located at the base of thehydrophobic cleft and forms direct interactions with residues of the CDRH3, knocks out binding when mutated to alanine possibly by destabilizingthe cleft itself.

FIG. 32 is a table listing neutralization IC50s and IC80s of 10E8alanine scanning variants. In cases where neutralization IC50 or IC80were not achieved at the highest concentration of antibody, the highestconcentration was used in calculation of the mean. ^(^)Mean fold effectis defined as the average of the individual fold effects seen againsteach viral strain. ^(&)Mutations Y99A_(HC), F100aA_(HC), W100bA_(HC),and G100hA_(HC) all had detrimental effects on neutralization,decreasing potency by over 1000-fold. Other mutations also had strongeffects on neutralization, including P100gA_(HC) and E100iA_(HC) of theCDR H3, and W33A_(HC) and R50A_(HC) within the hydrophobic cleft. Lightchain mutation R91A_(LC), similarly to its effects on binding topeptide, decreased neutralization potency by over 1000-fold.

FIG. 33 is a table illustrating root-mean-square deviations (RMSD) ofantibody-bound gp41 structures. Alignments were performed with the useof the program LSQKAB (Winn, M. D. et al. Acta Crystallogr D BiolCrystallogr, 67, 235-242, 2011). For the 4E10: 41 complex the structureof PDB ID No. 2FX7 was used (incorporated by reference herein as presentin the database on Oct. 22, 2012). For the Z13e1:gp41 complex thestructure of PDB ID No. 3FN0 was used (incorporated by reference hereinas present in the database on Oct. 22, 2012). For the 2F5: 41 complexthe structure of PDB ID No. 1TJI was used).

FIG. 34 is a table showing comparison of MPER-specific antibody buriedsurfaces on gp41. The interaction studies were performed with PISA(ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver). Values in parentheses arefor complex 2. For the 4E10:gp41 complex the structure of PDB ID No.2FX7 was used (incorporated by reference herein as present in thedatabase on Oct. 22, 2012). For the Z13e1:gp41 complex the structure ofPDB ID No. 3FNO was used (incorporated by reference herein as present inthe database on Oct. 22, 2012). For the 2F5:gp41 complex the structureof PDB ID No. 1TJI was used (incorporated by reference herein as presentin the database on Oct. 22, 2012). Despite recognizing a more extensiveresidue range, 10E8 has a smaller gp41 footprint than 4E10. If thecomparison is limited to the overlapping peptide range, residues671-683, the difference in the footprints is even more pronounced, with10E8 burying roughly 25% less surface area on gp41 than 4E10.

FIG. 35 is a table showing comparison of direct contacts of antibodies10E8 and 4E10 with gp41. Direct contacts were determined with the use ofthe program Ligplot (McDonald et al., J Mol Biol 238, 777-793, 1994). Hrefers to hydrogen bond; N refers to van der Waals contact.

FIG. 36 is a se t of tables illustrating results from the neutralizationof pseudotyped COT6.15 (clade C) envelope MPER mutants with 10E8 and4E10 antibodies.

FIG. 37 is a table illustrating results of neutralization assays usingthe cross-complementation of the 10E8, 7H6 and 7N16 heavy and lightchains.

FIG. 38 is a schematic diagram of the crystal structure of the antibody10E8 in complex with a gp41 peptide, showing the electrostatic surfacecharge of the antibody.

FIG. 39 is a schematic diagram of the crystal structure of the antibody10E8 in complex with a gp41 peptide, showing the electrostatic surfacecharge of the peptide.

FIG. 40 is a schematic diagram of the crystal structure of the antibody10E8 in complex with a gp41 peptide, showing the positions of theresidue changes in the 10E8 variant antibodies 7H6 and 7N16.

FIG. 41 is digital images of the crystal structures of the 2F5, 4E10 andZ13E1 antibodies in complex with gp41 peptides and a schematic of gp41illustrating the relative biding sites of the 2F5, 4E10, Z13E1 and 10E8antibodies. The amino acid sequence of SEQ ID NO: 26 is shown.

FIGS. 42A and 42B are scattergram plots showing the results of FACSisolation of CD19⁺IgA⁻IgD⁻IgM⁻ B cells from a PBMC sample.

FIG. 43 is a table and a set of ribbon diagrams illustrating an alaninescan of the indicated 10E8 residues (Kabat positions) as described inExamples 1 and 8.

FIGS. 44A and 44B are a series of ribbon diagrams illustrating thestructure-based mutagenesis of 10E8 to enhance hydrophobic interactionswith gp41.

FIG. 45 is a table illustrating the results of neutralization assays ona panel of HIV-1 viruses using the structure-base 10E8 mutants describedin FIGS. 44A and 44B and Example 8 (with reference to Kabat numbering).

FIG. 46 is a schematic diagram illustrating design of partially-revertedantibody variants.

FIG. 47 is a sequence alignment and a ribbon diagram illustratingpartial germline revertants of the 10E8 heavy and light chain. Thesequences shown are SEQ ID NO: 7 (10E8_germ_H), SEQ ID NO: 147(10E8gH01), SEQ ID NO: 148 (10E8gH02), SEQ ID NO: 149 (10E8gH03), SEQ IDNO: 1 (10E8_HEAVY), SEQ ID NO: 8 (10E8_germ_L), SEQ ID NO: 150(10E8gL01), SEQ ID NO: 151 (10E8gL02), SEQ ID NO: 152 (10E8gL03), SEQ IDNO: 2 (10E8—LIGHT).

FIG. 48 is a set of tables illustrating results of neutralization assayson a panel of HIV-1 viruses using the partial germline revertants of the10E8 antibody as described in FIG. 47 and Example 8. “10E8-R1” refers to10E8gH01 heavy chain (SEQ ID NO: 147) paired with 10E8gL01 light chain(SEQ ID NO: 150). “10E8-R3” refers to 10E8gH03 heavy chain (SEQ ID NO:149) paired with 10E8gL03 light chain (SEQ ID NO: 152).

FIG. 49 is a set of tables illustrating results of neutralization assayson a panel of HIV-1 viruses using a series of 10E8 mutants on the10E8gH03 heavy chain background (SEQ ID NO: 149) or the 10E8gL03background (SEQ ID NO: 152) as shown in FIG. 47 and described in Example8.

FIGS. 50A-50F are a series of charts illustrating identification ofsomatic variants of antibody 10E8 by deep sequencing and grid sampling.(A) 10E8-identity/divergence plots of donor N152 heavy-chain antibodyome(left), with grid sampling (right). Identity to 10E8 is shown on thevertical axis, and divergence from germline V-gene origin is plotted onthe horizontal axis, with frequency of antibodies shown as a heat map.Grid sampling is shown, with selected antibodies that either did notexpress or bind to MPER in hollow circles and with selected antibodiesthat did bind in solid circles, shaded according to their phylogeneticdistance from 10E8 in (C). (B) 10E8-identity/divergence plots of donorN152 light-chain antibodyome (left), with grid sampling (right). Axesand shading are the same as in (A). (C/D) Phylogenetic trees ofgrid-identified variant for heavy chain (C) and light chain (D). (E-F)Neutralization of 10E8 and 10E8 variants, assessed in duplicate on 6HIV-1 isolates for heavy chain variants (E) and light chain variants(F). The average IC50s of gVRC-H1_(dN152):10E8L andgVRC-H11_(dN152):10E8L was roughly 6-fold improved over the originaltemplate 10E8. Variants are arranged and named by their genetic distancefrom 10E8, and shaded relative to their phylogenetic distance.

FIGS. 51A-51D are a series of sequence alignments and ribbon diagramsillustrating sequences and modeled structures of 10E8 variants thatneutralize HIV-1. (A) Heavy-chain sequences (SEQ ID NOs: 1, and 153-163,descending; gVRC-H1_(dN152) (SEQ ID NO: 153); gVRC-H2_(dN152) (SEQ IDNO: 154); gVRC-H3_(dN152) (SEQ ID NO: 155); gVRC-H4_(dN152) (SEQ ID NO:156); gVRC-H5_(dN152) (SEQ ID NO: 157); gVRC-H6_(dN152) (SEQ ID NO:158); gVRC-H7_(dN152) (SEQ ID NO: 159); gVRC-H8_(dN152) (SEQ ID NO:160); gVRC-H9_(dN152) (SEQ ID NO: 161); gVRC-H10_(dN152) (SEQ ID NO:162) gVRC-H11_(dN152) (SEQ ID NO: 163)) Sequences are arranged bygenetic distance from 10E8, with sequence changes from 10E8 underlined.Framework and CDR residues are highlighted, as are residues thatinteract with the gp41MPER epitope (open circle, mainchain interaction;open circle with rays, sidechain interactions; solid circle, bothmainchain and sidechain interactions). (B) Modeled structures of heavychain variants in complex with gp41 epitope. 10E8 variants with enhancedrecognition (with heavy chains shaded according to phylogenetic distanceas in 1B) were modeled onto the structure of 10E8 with the entire MPERregion of HIV-1 gp41 (off-white). Structures are displayed asCα-ribbons, with amino acid-side chains that vary from 10E8 highlightedin stick representation, dark grey. (C) Light-chain sequences (SEQ IDNOs: 2 and 164-182, descending; gVRC-L1_(dN152) (SEQ ID NO: 164);gVRC-L2_(dN152) (SEQ ID NO: 165); gVRC-L3_(dN152) (SEQ ID NO: 166);gVRC-L4_(dN152) (SEQ ID NO: 167); gVRC-L5_(dN152) (SEQ ID NO: 168);gVRC-L6_(dN152) (SEQ ID NO: 169); gVRC-L7_(dN152) (SEQ ID NO: 170);gVRC-L8_(dN152) (SEQ ID NO: 171); gVRC-L9_(dN152) (SEQ ID NO: 172);gVRC-L10_(d152) (SEQ ID NO: 173); gVRC-L11_(dN152) (SEQ ID NO: 174);gVRC-L12_(dN152) (SEQ ID NO: 175); gVRC-L13_(dN152) (SEQ ID NO: 176);gVRC-L14_(dN152) (SEQ ID NO: 177); gVRC-L15_(dN152) (SEQ ID NO: 178);gVRC-L16_(dN152) (SEQ ID NO: 179); gVRC-L17_(dN152) (SEQ ID NO: 180);gVRC-L18_(dN152) (SEQ ID NO: 181); gVRC-L19_(dN152) (SEQ ID NO: 182);.Sequences are arranged by genetic distance from 10E8, with sequencechanges from 10E8 underlined. Framework and CDR residues arehighlighted, as are residues that interact with the gp41MPER epitope (asdescribed in (A). (D) Modeled structures of light chain variants incomplex with gp41 epitope. 10E8 variants with enhanced recognition (withlight chains shaded according to phylogenetic distance as in 1C) weremodeled onto the structure of 10E8 with entire MPER region of HIV-1 gp41(off-white). Structures are displayed as Cα-ribbons, with aminoacid-side chains that vary from 10E8 highlighted in stickrepresentation, dark grey.

FIGS. 52A-52D are a table and a set of graphs illustrating phylogeneticbranch matching of heavy and light chain variants of 10E8. (A)Phylogenetic branch matching. From the phylogenetic trees ofgrid-identified antibodies (FIG. 1 C,D) branches were named based ondistance from 10E8, b1-H for heavy and b1-L for light for the branchcontaining 10E8, and in descending order, b2-H (b2-L), b3-H (b3-L), andb4-H for the farthest branch. The variant from each branch thatdisplayed the most potent neutralization with a 10E8 wild-type partnerwas chosen, and a full matrix of 12 antibodies reconstituted. (B) HIV-1neutralization. Neutralization was assessed on 5 isolates, for 10E8variants from matched and mismatched branch pairings. (C) Hep2 StainingAuto-reactivity was accessed with Hep2 cell staining, for 10E8 variantsfrom matched and mismatched branch pairings.

FIG. 53 is a table illustrating 10E8 monoclonal antibodies composed ofthe indicated heavy and light chain variants based on the neutralizationpotency of the indicated variants when complemented with the 10E8wild-type complementary chain.

FIG. 54 is a table illustrating 10E8 antibodies composed of theindicated heavy and light chain variants paired phylogenetically.

FIGS. 55A-55C shows a set of tables illustrating results ofneutralization assays on a panel of HIV-1 viruses using the series of10E8 variant paired by neutralization potency or pair phylogeneticallyas shown in FIGS. 53 and 54 and described in Example 8.

FIG. 56 is a set of tables illustrating results of autoreactivity assayson a panel of HIV-1 viruses using the series of 10E8 variant paired byneutralization potency or pair phylogenetically as shown in FIGS. 53 and54 and described in Example 8.

FIG. 57 is a set of tables illustrating results of neutralization assayson a panel of HIV-1 viruses using the series of 10E8 variants paired byneutralization potency or paired phylogenetically as shown in FIGS. 53and 54 and described in Example 8.

FIGS. 58A and 58B are a set of graphs illustrating results fromneutralization assays testing the neutralization properties of theindicated series of antibodies, containing 10E8 heavy and light chains,or variants of 10E8 heavy and light chains, on a panel of 20 HIVviruses.

FIG. 59 is a table illustrating the nomenclature and SEQ ID NOs for theheavy and light chains of certain 10E8 antibody variants.

FIGS. 60A and 60B are a set of tables illustrating a summary of 10E8heavy chain variants. “Germline revertant,” “454,” “Alanine Scan,”“Structure-based,” and “additional” mutants refer to the 10E8 heavychain substitutions illustrated in Example 8.

FIGS. 61A and 61B are a set of tables illustrating a summary of 10E8light chain variants. “Germline revertant” and “454” mutants refer tothe 10E8 light chain substitutions illustrated in Example 8.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile in the form of the file named “Sequence.txt” (˜160 kb), which wascreated on Apr. 22, 2014, which is incorporated by reference herein. Inthe accompanying sequence listing:

SEQ ID NO: 1 is the amino acid sequence of the heavy chain of thegp41-specific antibody 10E8.

SEQ ID NO: 2 is the amino acid sequence of the light chain of thegp41-specific antibody 10E8.

SEQ ID NO: 3 is the amino acid sequence of the heavy chain of the41-specific antibody 7H6.

SEQ ID NO: 4 is the amino acid sequence of the light chain of thegp41-specific antibody 7H6.

SEQ ID NO: 5 is the amino acid sequence of the heavy chain of thegp41-specific antibody 7N16.

SEQ ID NO: 6 is the amino acid sequence of the light chain of thegp41-specific antibody 7N16.

SEQ ID NO: 7 is the amino acid sequence of the heavy chain ofIGHV3-15*05.

SEQ ID NO: 8 is the amino acid sequence of the light chain ofIGLV3-19*01.

SEQ ID NOs: 9 and 10 are epitopes within the gp41MPER.

SEQ ID NO: 11 is the amino acid sequence of the heavy chain of agp41-specific antibody.

SEQ ID NO: 12 is the amino acid sequence of the light chain of agp41-specific antibody.

SEQ ID NO: 13 is the amino acid sequence of a gp41 epitope thatspecifically binds 10E8 and 10E8 like antibodies.

SEQ ID NOs: 14-25 are the amino acid sequences of mutant gp41MPERsequences.

SEQ ID NO: 26 is the amino acid sequence of a region of the gp41MPER.

SEQ ID NOs: 27-34 are the nucleic acid sequences of sequencing primers.

SEQ ID NOs: 35-115 are the nucleic acid sequences of exemplary 10E8-likeantibody heavy chains

SEQ ID NOs: 116-145 are the nucleic acid sequences of exemplary10E8-like antibody light chains.

SEQ ID NO: 146 is the consensus amino acid sequence of the heavy chainof a gp41-specific antibody.

SEQ ID NOs: 147-149 are the amino acid sequences of the heavy chains ofgermline revertants the 10E8 monoclonal antibody.

SEQ ID NOs: 150-152 are the amino acid sequences of the light chainvariable regions of germline revertants of the 10E8 monoclonal antibody.

SEQ ID NOs: 153-163 are the amino acid sequences of the heavy chainvariable regions of gp41-specific antibodies.

SEQ ID NOs: 164-186 are the amino acid sequences of the light chainvariable regions of gp41-specific antibodies.

SEQ ID NO: 187 is the consensus amino acid sequence of the heavy chainvariable region of a gp41-specific antibody.

SEQ ID NO: 188 is the consensus amino acid sequence of the heavy chainvariable region of a gp41-specific antibody.

SEQ ID NOs: 189-192 are the amino acid sequences of the heavy chainvariable regions of gp41-specific antibodies.

SEQ ID NOs: 193-196 are the nucleic acid sequences of primers.

SEQ ID NOs: 197-199 are the amino acid sequences of the light chainvariable regions of gp41 specific antibodies.

SEQ ID NOs: 200-205 are the amino acid sequences of the heavy chainvariable regions of gp41 specific antibodies.

SEQ ID NOs: 205-209 are the amino acid sequences of the light chainvariable regions of gp41 specific antibodies.

DETAILED DESCRIPTION

I. Summary of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology canbe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 1999; Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995; and other similarreferences.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “an antigen” includes single or pluralantigens and can be considered equivalent to the phrase “at least oneantigen.”

As used herein, the term “comprises” means “includes.” Thus, “comprisingan antigen” means “including an antigen” without excluding otherelements.

It is further to be understood that any and all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescriptive purposes, unless otherwise indicated. Although many methodsand materials similar or equivalent to those described herein can beused, particular suitable methods and materials are described below. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

To facilitate review of the various embodiments, the followingexplanations of terms are provided:

Administration: The introduction of a composition into a subject by achosen route. Administration can be local or systemic. For example, ifthe chosen route is intravenous, the composition is administered byintroducing the composition into a vein of the subject. In someexamples, a disclosed antibody, or antigen binding fragment thereof,specific for an HIV gp41 polypeptide is administered to a subject.

Agent: Any substance or any combination of substances that is useful forachieving an end or result; for example, a substance or combination ofsubstances useful for inhibiting HIV infection in a subject. Agentsinclude proteins, nucleic acid molecules, compounds, small molecules,organic compounds, inorganic compounds, or other molecules of interest.An agent can include a therapeutic agent (such as an anti-retroviralagent), a diagnostic agent or a pharmaceutical agent. In someembodiments, the agent is a polypeptide agent (such as aHIV-neutralizing antibody), or an anti-viral agent. The skilled artisanwill understand that particular agents may be useful to achieve morethan one result.

Amino acid substitution: The replacement of one amino acid inpolypeptide with a different amino acid.

Amplification: A technique that increases the number of copies of anucleic acid molecule (such as an RNA or DNA). An example ofamplification is the polymerase chain reaction, in which a biologicalsample is contacted with a pair of oligonucleotide primers, underconditions that allow for the hybridization of the primers to a nucleicacid template in the sample. The primers are extended under suitableconditions, dissociated from the template, and then re-annealed,extended, and dissociated to amplify the number of copies of the nucleicacid. The product of amplification can be characterized byelectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in WO 90/01069; ligase chain reaction amplification, asdisclosed in EP-A-320 308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: A polypeptide substantially encoded by an immunoglobulin geneor immunoglobulin genes, or antigen binding fragments thereof, whichspecifically binds and recognizes an analyte (antigen) such as gp41 oran immunogenic fragment of gp41, for example the membrane-proximalregion of gp41. Immunoglobulin genes include the kappa, lambda, alpha,gamma, delta, epsilon and mu constant region genes, as well as themyriad immunoglobulin variable region genes.

Antibodies exist, for example as intact immunoglobulins and as a numberof well characterized antigen binding fragments produced by digestionwith various peptidases. For instance, Fabs, Fvs, scFvs thatspecifically bind to gp41 or fragments of gp41 would be gp41-specificbinding agents. A scFv protein is a fusion protein in which a lightchain variable region of an immunoglobulin and a heavy chain variableregion of an immunoglobulin are bound by a linker, while in dsFvs, thechains have been mutated to introduce a disulfide bond to stabilize theassociation of the chains. The term also includes genetically engineeredforms such as chimeric antibodies and heteroconjugate antibodies such asbispecific antibodies. See also, Pierce Catalog and Handbook, 1994-1995(Pierce Chemical Co., Rockford, Ill.); Kuby, Immunology, 3^(rd) Ed.,W.H. Freeman & Co., New York, 1997.

Antibody fragments include, but are not limited to, the following: (1)Fab, the fragment which contains a monovalent antigen-binding fragmentof an antibody molecule produced by digestion of whole antibody with theenzyme papain to yield an intact light chain and a portion of one heavychain; (2) Fab′, the fragment of an antibody molecule obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule; (3) (Fab′)₂, the fragment of theantibody obtained by treating whole antibody with the enzyme pepsinwithout subsequent reduction; (4) F(ab′)₂, a dimer of two Fab′ fragmentsheld together by two disulfide bonds; (5) Fv, a genetically engineeredfragment containing the variable region of the light chain and thevariable region of the heavy chain expressed as two chains; and (6)single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain, the variable regionof the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

The term “antibody,” as used herein, also includes antibody fragmentseither produced by the modification of whole antibodies or thosesynthesized de novo using recombinant DNA methodologies. In someexamples, the term antibody includes the amino acid sequences of one ormore of the CDRs from the antibody grafted onto a scaffold.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (κ). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE. The disclosedantibodies can be class switched.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In severalembodiments, the heavy and the light chain variable domains combine tospecifically bind the antigen. In additional embodiments, only the heavychain variable domain is required. For example, naturally occurringcamelid antibodies consisting of a heavy chain only are functional andstable in the absence of light chain (see, e.g., Hamers-Casterman etal., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol.,3:733-736, 1996). Light and heavy chain variable domains contain a“framework” region interrupted by three hypervariable regions, alsocalled “complementarity-determining regions” or “CDRs” (see, e.g., Kabatet al., Sequences of Proteins of Immunological Interest, U.S. Departmentof Health and Human Services, 1991). The sequences of the frameworkregions of different light or heavy chains are relatively conservedwithin a species. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The amino acid sequence boundaries of a given CDR can bereadily determined using any of a number of well-known schemes,including those described by Kabat et al. (“Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991; “Kabat” numbering scheme),Al-Lazikani et al., (JMB 273,927-948, 1997; “Chothia” numbering scheme),and Lefranc, et al. (“IMGT unique numbering for immunoglobulin and Tcell receptor variable domains and Ig superfamily V-like domains,” Dev.Comp. Immunol., 27:55-77, 2003; “IMGT” numbering scheme).

The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3(from the N-terminus to C-terminus), and are also typically identifiedby the chain in which the particular CDR is located. Thus, a V_(H) CDR3is located in the variable domain of the heavy chain of the antibody inwhich it is found, whereas a V_(L) CDR1 is the CDR1 from the variabledomain of the light chain of the antibody in which it is found. Lightchain CDRs are sometimes referred to as CDR L1, CDR L2, and CDR L3.Heavy chain CDRs are sometimes referred to as CDR H1, CDR H2, and CDRH3.

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an antibody fragment, suchas Fv, scFv, dsFv or Fab. References to “V_(L)” or “VL” refer to thevariable region of an immunoglobulin light chain, including that of anFv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. These fused cells and their progeny are termed“hybridomas.” Monoclonal antibodies include humanized monoclonalantibodies. In some examples monoclonal antibodies are isolated from asubject. The amino acid sequences of such isolated monoclonal antibodiescan be determined.

A “humanized” immunoglobulin is an immunoglobulin including a humanframework region and one or more CDRs from a non-human (such as a mouse,rat, or synthetic) immunoglobulin. The non-human immunoglobulinproviding the CDRs is termed a “donor,” and the human immunoglobulinproviding the framework is termed an “acceptor.” In one embodiment, allthe CDRs are from the donor immunoglobulin in a humanizedimmunoglobulin. Constant regions need not be present, but if they are,they must be substantially identical to human immunoglobulin constantregions, such as at least about 85-90%, such as about 95% or moreidentical. Hence, all parts of a humanized immunoglobulin, exceptpossibly the CDRs, are substantially identical to corresponding parts ofnatural human immunoglobulin sequences. A “humanized antibody” is anantibody including a humanized light chain and a humanized heavy chainimmunoglobulin. A humanized antibody binds to the same antigen as thedonor antibody that provides the CDRs. The acceptor framework of ahumanized immunoglobulin or antibody may have a limited number ofsubstitutions by amino acids taken from the donor framework. Humanizedor other monoclonal antibodies can have additional conservative aminoacid substitutions which have substantially no effect on antigen bindingor other immunoglobulin functions. Humanized immunoglobulins can beconstructed by means of genetic engineering (for example, see U.S. Pat.No. 5,585,089).

Antibody self-reactivity: A property of an antibody, whereby theantibody reacts with self-epitopes, that is epitopes of proteins and/orlipids that are produced by the subject. For example, an antibody, suchas 10E8 that does not have self-reactivity does not bind to lipidspresent on the membrane of a cell from a subject, such as a cellinfected with HIV and/or expressing gp41 on its surface. Methods ofdetermining if an antibody reacts with self epitopes are known to theperson of ordinary skill in the art and described herein (for example,in Examples 1 and 8). In one example, antibody self reactivity isevaluated using an anti-cardiolipin assay or an anti-nuclear antigen(ANA) assay.

Antibody Scaffold: Refers to a heterologous protein that is engraftedwith one or more CDRs from an antibody of interest on its surface.Transplantation of the CDRs can performed computationally in a mannerthat preserves its relevant structure and conformation. Mutations withinthe acceptor scaffold are made in order to accommodate the CDR graft.

Antibodyome: The entire repertoire of expressed antibody heavy and lightchain sequence in an individual. The individual can be an individualinfected with a pathogen, for example, HIV.

Antigen: A polypeptide that can stimulate the production of antibodiesor a T cell response in an animal, including polypeptides that areinjected or absorbed into an animal. An antigen reacts with the productsof specific humoral or cellular immunity, including those induced byheterologous antigens, such as the disclosed antigens. “Epitope” or“antigenic determinant” refers to the region of an antigen to which Band/or T cells respond. In one embodiment, T cells respond to theepitope, when the epitope is presented in conjunction with an MHCmolecule. Epitopes can be formed both from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of a protein.Epitopes formed from contiguous amino acids are typically retained onexposure to denaturing solvents whereas epitopes formed by tertiaryfolding are typically lost on treatment with denaturing solvents. Anepitope typically includes at least 3, and more usually, at least 5,about 9, or about 8-10 amino acids in a unique spatial conformation.Methods of determining spatial conformation of epitopes include, forexample, x-ray crystallography and nuclear magnetic resonance.

Immunogenic polypeptides and immunogenic peptides are non-limitingexamples of antigens. In some examples, antigens include polypeptidesderived from a pathogen of interest, such as a virus. An antigen thatcan stimulate the production of antibodies or a T cell response in asubject to a polypeptide expressed by a virus is a viral antigen. An“HIV antigen” can stimulate the production of antibodies or a T cellresponse in a subject to a polypeptide expressed by HIV. In someembodiments, an HIV antigen is a polypeptide expressed by HIV, such asgp160, or a fragment thereof, such as gp145, gp140, gp120 or gp41.

A “target epitope” is a specific epitope on an antigen that specificallybinds an antibody of interest, such as a monoclonal antibody. In someexamples, a target epitope includes the amino acid residues that contactthe antibody of interest, such that the target epitope can be selectedby the amino acid residues determined to be in contact with the antibodyof interest.

Anti-retroviral agent: An agent that specifically inhibits a retrovirusfrom replicating or infecting cells. Non-limiting examples ofantiretroviral drugs include entry inhibitors (e.g., enfuvirtide), CCR5receptor antagonists (e.g., aplaviroc, vicriviroc, maraviroc), reversetranscriptase inhibitors (e.g., lamivudine, zidovudine, abacavir,tenofovir, emtricitabine, efavirenz), protease inhibitors (e.g.,lopivar, ritonavir, raltegravir, darunavir, atazanavir), maturationinhibitors (e.g., alpha interferon, bevirimat and vivecon).

Anti-retroviral therapy (ART): A therapeutic treatment for HIV infectioninvolving administration of at least one anti-retroviral agents (e.g.,one, two, three or four anti-retroviral agents) to an HIV infectedindividual during a course of treatment. Non-limiting examples ofantiretroviral agents include entry inhibitors (e.g., enfuvirtide), CCR5receptor antagonists (e.g., aplaviroc, vicriviroc, maraviroc), reversetranscriptase inhibitors (e.g., lamivudine, zidovudine, abacavir,tenofovir, emtricitabine, efavirenz), protease inhibitors (e.g.,lopivar, ritonavir, raltegravir, darunavir, atazanavir), maturationinhibitors (e.g., alpha interferon, bevirimat and vivecon). One exampleof an ART regimen includes treatment with a combination of tenofovir,emtricitabine and efavirenz. In some examples, ART includes HighlyActive Anti-Retroviral Therapy (HAART).

Atomic Coordinates or Structure coordinates: Mathematical coordinatesderived from mathematical equations related to the patterns obtained ondiffraction of a monochromatic beam of X-rays by the atoms (scatteringcenters) such as an antigen, or an antigen in complex with an antibody.In some examples that antigen can be gp41, a gp41:antibody complex, orcombinations thereof in a crystal. The diffraction data are used tocalculate an electron density map of the repeating unit of the crystal.The electron density maps are used to establish the positions of theindividual atoms within the unit cell of the crystal. In one example,the term “structure coordinates” refers to Cartesian coordinates derivedfrom mathematical equations related to the patterns obtained ondiffraction of a monochromatic beam of X-rays, such as by the atoms of agp41 in crystal form.

Those of ordinary skill in the art understand that a set of structurecoordinates determined by X-ray crystallography is not without standarderror. For the purpose of this disclosure, any set of structurecoordinates that have a root mean square deviation of protein backboneatoms (N, Cα, C and O) of less than about 1.0 Angstroms whensuperimposed, such as about 0.75, or about 0.5, or about 0.25 Angstroms,using backbone atoms, shall (in the absence of an explicit statement tothe contrary) be considered identical.

B Cell and Memory B cell: B cells are a subset of lymphocytes, that is,white blood cells (leukocytes). Mature B cells differentiate into plasmacells, which produces antibodies, and memory B cells. A “B cellprogenitor” is a cell that can develop into a mature B cell. B cellprogenitors include stem cells, early pro-B cells, late pro-B cells,large pre-B cells, small pre-B cells, and immature B cells andtransitional B cells. Generally, early pro-B cells (that express, forexample, CD43 or B220) undergo immunoglobulin heavy chain rearrangementto become late pro B and pre B cells, and further undergo immunoglobulinlight chain rearrangement to become an immature B cells. In humans,immature B cells (for example, immature peripheral transitional B cells)include CD38^(hi), IgD⁺, CD10⁺, CD24^(hi), CD44^(lo), CD23^(lo) andCD1^(lo) cells. Thus, immature B cells include B220 (CD45R) expressingcells wherein the light and the heavy chain immunoglobulin genes arerearranged. In one embodiment, immature B cells express CD45R, class II,IgM, CD19 and CD40. Immature B cells can develop into mature B cells,which can produce immunoglobulins (e.g., IgA, IgG or IgM). Mature Bcells have acquired surface IgM and IgD, are capable of responding toantigen, and express characteristic markers such as CD21 and CD23(CD23^(hi)CD21^(hi) cells). Plasma cells are terminally differentiated Bcells that are the predominant antibody-secreting cells.

After a B cell progenitor (e.g., a pre-committed small lymphocyte) isstimulated by an antigen, it differentiates into a blast cell, whichdifferentiates into an immature plasma cell that can differentiate intoeither a mature plasma cell or a memory B cell. A “mature plasma cell”secretes immunoglobulins in response to a specific antigen.

B cells can be activated by agents such as lippopolysaccharide (LPS) orIL-4 and antibodies to IgM. Common biological sources of B cells and Bcell progenitors include bone marrow, peripheral blood, spleen and lymphnodes.

A “memory B cell” is a B cell that undergoes isotype switching andsomatic hypermutation that are generally found during a secondary immuneresponse (a subsequent antigen exposure following a primary exposure)but can also be detected during a primary antigen response. Generationof memory B cells generally requires helper T cells. The development ofmemory B cells takes place in germinal centers (GC) of lymphoidfollicles where antigen-driven lymphocytes undergo somatic hypermutationand affinity selection, presumably under the influence of helper Tcells. Typically, memory B cells express high affinity antigen specificimmunoglobulin (B cell receptor) on their cell surface. In oneembodiment, memory B cells include cells that express CD19, but do notexpress IgA, IgD or IgM (CD19⁺IgA⁻IgD⁻IgM⁻ cells).

Bispecific antibody: A recombinant molecule composed of two differentantigen binding domains that consequently binds to two differentantigenic epitopes. Bispecific antibodies include chemically orgenetically linked molecules of two antigen-binding domains. The antigenbinding domains can be linked using a linker. The antigen bindingdomains can be monoclonal antibodies, antigen-binding fragments (e.g.,Fab, scFv), eAds, bispecific single chain antibodies or combinationsthereof. A bispecific antibody can include one or more constant domains,but does not necessarily include a constant domain. An example of abispecific antibody is a bispecific single chain antibody including ascFv that specifically binds to gp41 joined (via a peptide linker) to ascFv that specifically binds to an antigen other than gp41. Anotherexample is a bispecific antibody including a Fab that specifically bindsto gp41 joined to a scFv that specifically binds to an antigen otherthan gp41.

B cell repertoire: The B cells in a sample or in a subject specific forantigen of interest.

CD40: A costimulatory protein found an antigen presenting cells that isrequired for their activation. The binding of CD40 ligand (CD40L), alsoknown as CD154, to CD40 activates antigen presenting cells. Thisreceptor has been found to be essential in mediating a broad variety ofimmune and inflammatory responses including T cell-dependentimmunoglobulin class switching, memory B cell development, and germinalcenter formation. An exemplary amino acid sequence for CD40, and anexemplary mRNA sequence encoding CD40 can be found in GENBANK® AccessionNo. NM_001250, (Jun. 10, 2012), which is incorporated herein byreference.

CD40 ligand (CD40L): A ligand that is also called CD154, that isexpressed on activated T cells and is a member of the tumor necrosissuperfamily of molecules. It binds to CD40 on antigen-presenting cells,which leads to many effects depending on the target cell type. Ingeneral, CD40L plays the role of a costimulatory molecule and inducesactivation in antigen-presenting cells in association with T cellreceptor stimulation by MHC molecules on the antigen-presenting cells.In total CD40L has three binding partners: CD40, α5β1 integrin andαIIbβ3. CD154 is expressed on the surface of T cells. It regulates Bcell function by engaging CD40 on the B cell surface. An exemplary aminoacid sequence for CD40, and an exemplary mRNA sequence encoding CD40 canbe found in GENBANK® Accession No. NM_000074.2, (Jun. 10, 2012), whichis incorporated herein by reference.

Chimeric antibody: An antibody which includes sequences derived from twodifferent antibodies, which typically are of different species. In someexamples, a chimeric antibody includes one or more CDRs and/or frameworkregions from one human antibody and CDRs and/or framework regions fromanother human antibody. In some examples, a chimeric antibody isproduced by grafting one or more CDRs into an antibody scaffold.

Clonal variant: Any sequence, which differs by one or more nucleotidesor amino acids, in presence of V region with identical mutationscompared to the germline, identical VDJ or VJ gene usage, and identicalD and J length. The “germline” sequence is intended to be the sequencecoding for the antibody/immunoglobulin (or of any fragment thereof)deprived of mutations, for example somatic mutations. The percentage ofhomology represents an indication of the mutational events which anytype of heavy chain portion undergoes after contact with an antigen.

Computer readable storage media: Any medium or media, which can be readand accessed directly by a computer, so that the media is suitable foruse in a computer system. Such media include, but are not limited to:magnetic storage media such as floppy discs, hard disc storage mediumand magnetic tape; optical storage media such as optical discs orCD-ROM; electrical storage media such as RAM and ROM; and hybrids ofthese categories such as magnetic/optical storage media.

Computer system: Hardware that can be used to analyze atomic coordinatedata and/or design an antigen using atomic coordinate data or to analyzean amino acid or nucleic acid sequence, for example to compare two ormore sequences an calculate sequence similarity and/or divergence. Theminimum hardware of a computer-based system typically includes a centralprocessing unit (CPU), an input device, for example a mouse, keyboard,and the like, an output device, and a data storage device. Desirably amonitor is provided to visualize structure data. The data storage devicemay be RAM or other means for accessing computer readable. Examples ofsuch systems are microcomputer workstations available from SiliconGraphics Incorporated and Sun Microsystems running Unix based Windows NTor IBM OS/2 operating systems.

Conjugate: A complex of two molecules linked together, for example,linked together by a covalent bond. In one embodiment, an antibody islinked to an effector molecule; for example, an antibody thatspecifically binds to gp41 covalently linked to an effector molecule orto a toxin. The linkage can be by chemical or recombinant means. In oneembodiment, the linkage is chemical, wherein a reaction between theantibody moiety and the effector molecule has produced a covalent bondformed between the two molecules to form one molecule. A peptide linker(short peptide sequence) can optionally be included between the antibodyand the effector molecule. Because conjugates can be prepared from twomolecules with separate functionalities, such as an antibody and aneffector molecule, they are also sometimes referred to as “chimericmolecules.” In one embodiment, an antibody linked to an effectormolecule is further joined to a lipid or other molecule to a protein orpeptide to increase its half-life in the body.

Contacting: Placement in direct physical association; includes both insolid and liquid form, which can take place either in vivo or in vitro.Contacting includes contact between one molecule and another molecule,for example the amino acid on the surface of one polypeptide, such as anantigen, that contacts another polypeptide, such as an antibody.Contacting between polypeptides can include direct contacts betweenamino acids of two or more polypeptides (for example, hydrogen bondingor Van der Waals force interactions between polypeptides), as well asother interactions between polypeptides producing an interface betweenthe polypeptides with reduced solvent accessibility (without all aminoacids of the interface necessarily forming direct bonds). In someembodiments, a direct contact refers to forming a hydrogen bond or Vander Waals interaction with particularly a identified residue or residuesin a sequence but not with the other residues in the sequence. Theperson of ordinary skill in the art is familiar with methods ofdetermining contacts between polypeptides (see for example, Example 1).Contacting can also include contacting a cell for example by placing anantibody in direct physical association with a cell. In someembodiments, an antibody (e.g., 10E8) only contacts particular residuesof an epitope on an antigen, such as the 10E8 epitope on gp41 asdescribed herein.

Control: A reference standard. In some embodiments, the control is asample obtained from a healthy patient. In other embodiments, thecontrol is a tissue sample obtained from a patient diagnosed with HIVinfection. In still other embodiments, the control is a historicalcontrol or standard reference value or range of values (such as apreviously tested control sample, such as a group of HIV patients withknown prognosis or outcome, or group of samples that represent baselineor normal values).

A difference between a test sample and a control can be an increase orconversely a decrease. The difference can be a qualitative difference ora quantitative difference, for example a statistically significantdifference. In some examples, a difference is an increase or decrease,relative to a control, of at least about 5%, such as at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, at least about 500%, or greater than 500%.

Cytokine/Interleukin (IL): A generic name for a diverse group of solubleproteins and peptides which act as humoral regulators at nano- topicomolar concentrations and which, either under normal or pathologicalconditions, modulate the functional activities of individual cells andtissues. These proteins also mediate interactions between cells directlyand regulate processes taking place in the extracellular environment.Many growth factors and cytokines act as cellular survival factors bypreventing programmed cell death. Cytokines and interleukins includeboth naturally occurring peptides and variants that retain full orpartial biological activity. Although specific cytokines/interleukinsare described in the specification, they are not limited to thespecifically disclosed peptides.

Cytotoxicity: The toxicity of a molecule, such as an immunotoxin, to thecells intended to be targeted, as opposed to the cells of the rest of anorganism. In one embodiment, in contrast, the term “toxicity” refers totoxicity of an immunotoxin to cells other than those that are the cellsintended to be targeted by the targeting moiety of the immunotoxin, andthe term “animal toxicity” refers to toxicity of the immunotoxin to ananimal by toxicity of the immunotoxin to cells other than those intendedto be targeted by the immunotoxin.

Detectable marker: A detectable molecule (also known as a label) that isconjugated directly or indirectly to a second molecule, such as anantibody, to facilitate detection of the second molecule. For example,the detectable marker can be capable of detection by ELISA,spectrophotometry, flow cytometry, microscopy or diagnostic imagingtechniques (such as CT scans, MRIs, ultrasound, fiberoptic examination,and laparoscopic examination). Specific, non-limiting examples ofdetectable markers include fluorophores, fluorescent proteins,chemiluminescent agents, enzymatic linkages, radioactive isotopes andheavy metals or compounds (for example super paramagnetic iron oxidenanocrystals for detection by MRI). In one example, a “labeled antibody”refers to incorporation of another molecule in the antibody. Forexample, the label is a detectable marker, such as the incorporation ofa radiolabeled amino acid or attachment to a polypeptide of biotinylmoieties that can be detected by marked avidin (for example,streptavidin containing a fluorescent marker or enzymatic activity thatcan be detected by optical or colorimetric methods). Various methods oflabeling polypeptides and glycoproteins are known in the art and may beused. Examples of labels for polypeptides include, but are not limitedto, the following: radioisotopes or radionuclides (such as ³⁵S or ¹³¹I),fluorescent labels (such as fluorescein isothiocyanate (FITC),rhodamine, lanthanide phosphors), enzymatic labels (such as horseradishperoxidase, beta-galactosidase, luciferase, alkaline phosphatase),chemiluminescent markers, biotinyl groups, predetermined polypeptideepitopes recognized by a secondary reporter (such as a leucine zipperpair sequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags), or magnetic agents, such as gadolinium chelates.In some embodiments, labels are attached by spacer arms of variouslengths to reduce potential steric hindrance. Methods for usingdetectable markers and guidance in the choice of detectable markersappropriate for various purposes are discussed for example in Sambrooket al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y., 1989) and Ausubel et al. (In Current Protocols in MolecularBiology, John Wiley & Sons, New York, 1998).

Detecting: To identify the existence, presence, or fact of something.General methods of detecting are known to the skilled artisan and may besupplemented with the protocols and reagents disclosed herein. Forexample, included herein are methods of detecting a cell that expressesgp41 in a subject.

DNA sequencing: The process of determining the nucleotide order of agiven DNA molecule. The general characteristics of “deep sequencing” arethat genetic material is amplified, such as by polymerase chainreaction, and then the amplified products are ligated to a solidsurface. The sequence of the amplified target genetic material is thenperformed in parallel and the sequence information is captured by acomputer. Generally, the sequencing can be performed using automatedSanger sequencing (AB 13730×1 genome analyzer), pyrosequencing on asolid support (454 sequencing, Roche), sequencing-by-synthesis withreversible terminations (ILLUMINA® Genome Analyzer),sequencing-by-ligation (ABI SOLiD®) or sequencing-by-synthesis withvirtual terminators (HELISCOPE®).

In some embodiments, DNA sequencing is performed using a chaintermination method developed by Frederick Sanger, and thus termed“Sanger based sequencing” or “SBS.” This technique usessequence-specific termination of a DNA synthesis reaction using modifiednucleotide substrates. Extension is initiated at a specific site on thetemplate DNA by using a short oligonucleotide primer complementary tothe template at that region. The oligonucleotide primer is extendedusing DNA polymerase in the presence of the four deoxynucleotide bases(DNA building blocks), along with a low concentration of a chainterminating nucleotide (most commonly a di-deoxynucleotide). Limitedincorporation of the chain terminating nucleotide by the DNA polymeraseresults in a series of related DNA fragments that are terminated only atpositions where that particular nucleotide is present. The fragments arethen size-separated by electrophoresis a polyacrylamide gel, or in anarrow glass tube (capillary) filled with a viscous polymer. Analternative to using a labeled primer is to use labeled terminatorsinstead; this method is commonly called “dye terminator sequencing.”

“Pyrosequencing” is an array based method, which has been commercializedby 454 Life Sciences (Branford, Conn.). In some embodiments of thearray-based methods, single-stranded DNA is annealed to beads andamplified via EmPCR®. These DNA-bound beads are then placed into wellson a fiber-optic chip along with enzymes that produce light in thepresence of ATP. When free nucleotides are washed over this chip, lightis produced as the PCR amplification occurs and ATP is generated whennucleotides join with their complementary base pairs. Addition of one(or more) nucleotide(s) results in a reaction that generates a lightsignal that is recorded, such as by the charge coupled device (CCD)camera, within the instrument. The signal strength is proportional tothe number of nucleotides, for example, homopolymer stretches,incorporated in a single nucleotide flow.

Effective amount: The amount of an agent (such as CD40L, IL-21 or IL-2)that alone, or together with one or more additional agents, induces thedesired response.

Effector molecule: The portion of a chimeric molecule that is intendedto have a desired effect on a cell to which the chimeric molecule istargeted. Effector molecule is also known as an effector moiety,therapeutic agent, or diagnostic agent, or similar terms.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, such that theyelicit a specific immune response, for example, an epitope is the regionof an antigen to which B and/or T cells respond. In some examples, adisclosed antibody specifically binds to an epitope on the surface ofgp41 from HIV.

Epitopes can be formed both from contiguous amino acids or noncontiguousamino acids juxtaposed by tertiary folding of a protein. Epitopes formedfrom contiguous amino acids are typically retained on exposure todenaturing solvents whereas epitopes formed by tertiary folding aretypically lost on treatment with denaturing solvents. An epitopetypically includes at least 3, and more usually, at least 5, about 9, orabout 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and nuclear magnetic resonance.

Fc polypeptide: The polypeptide including the constant region of anantibody excluding the first constant region immunoglobulin domain. Fcregion generally refers to the last two constant region immunoglobulindomains of IgA, IgD, and IgG, and the last three constant regionimmunoglobulin domains of IgE and IgM. An Fc region may also includepart or all of the flexible hinge N-terminal to these domains. For IgAand IgM, an Fc region may or may not include the tailpiece, and may ormay not be bound by the J chain. For IgG, the Fc region includesimmunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the lowerpart of the hinge between Cgamma1 (Cγ1) and Cγ2. Although the boundariesof the Fc region may vary, the human IgG heavy chain Fc region isusually defined to include residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat. For IgA, the Fc region includes immunoglobulin domains Calpha2and Calpha3 (Cα2 and Cα3) and the lower part of the hinge betweenCalpha1 (Cα1) and Cα2. Encompassed within the definition of the Fcregion are functionally equivalent analogs and variants of the Fcregion. A functionally equivalent analog of the Fc region may be avariant Fc region, including one or more amino acid modificationsrelative to the wild-type or naturally existing Fc region. Variant Fcregions will possess at least 50% homology with a naturally existing Fcregion, such as about 80%, and about 90%, or at least about 95%homology. Functionally equivalent analogs of the Fc region may includeone or more amino acid residues added to or deleted from the N- orC-termini of the protein, such as no more than 30 or no more than 10additions and/or deletions. Functionally equivalent analogs of the Fcregion include Fc regions operably linked to a fusion partner.Functionally equivalent analogs of the Fc region must include themajority of all of the Ig domains that compose the Fc region as definedabove; for example IgG and IgA Fc regions as defined herein must includethe majority of the sequence encoding CH₂ and the majority of thesequence encoding CH₃. Thus, the CH₂ domain on its own, or the CH₃domain on its own, are not considered the Fc region. The Fc region mayrefer to this region in isolation, or this region in the context of anFc fusion polypeptide (immunoadhesin, see below).

Framework Region: Amino acid sequences interposed between CDRs. Includesvariable light and variable heavy framework regions. The frameworkregions serve to hold the CDRs in an appropriate orientation for antigenbinding.

gp41: A specific HIV protein. The envelope protein of HIV-1 is initiallysynthesized as a longer precursor protein of 845-870 amino acids insize, designated gp160. gp160 forms a homotrimer and undergoesglycosylation within the Golgi apparatus. In vivo, it is then cleaved bya cellular protease into gp120 and gp41. The amino acid sequence of anexample of gp41 is set forth in GENBANK® Accession No. CAD20975 (asavailable on Oct. 16, 2009) which is incorporated by reference herein.It is understood that the sequence of gp41 can vary from that given inGENBANK® Accession No. CAD20975. gp41 contains a transmembrane domainand typically remains in a trimeric configuration; it interacts withgp120 in a non-covalent manner.

HIV Envelope protein (Env): The HIV envelope protein is initiallysynthesized as a longer precursor protein of 845-870 amino acids insize, designated gp160. gp160 forms a homotrimer and undergoesglycosylation within the Golgi apparatus. In vivo, it is then cleaved bya cellular protease into gp120 and gp41. gp120 contains most of theexternal, surface-exposed, domains of the HIV envelope glycoproteincomplex, and it is gp120 which binds both to cellular CD4 receptors andto cellular chemokine receptors (such as CCR5). gp41 contains atransmembrane domain and remains in a trimeric configuration; itinteracts with gp120 in a non-covalent manner.

Host cells: Cells in which a vector can be propagated and its DNAexpressed, for example a disclosed antibody can be expressed in a hostcell. The cell may be prokaryotic or eukaryotic. The term also includesany progeny of the subject host cell. It is understood that all progenymay not be identical to the parental cell since there may be mutationsthat occur during replication. However, such progeny are included whenthe term “host cell” is used.

Human Immunodeficiency Virus (HIV): A retrovirus that causesimmunosuppression in humans (HIV disease), and leads to a diseasecomplex known as the acquired immunodeficiency syndrome (AIDS). “HIVdisease” refers to a well-recognized constellation of signs and symptoms(including the development of opportunistic infections) in persons whoare infected by an HIV virus, as determined by antibody or western blotstudies. Laboratory findings associated with this disease include aprogressive decline in T cells. HIV includes HIV type 1 (HIV-1) and HIVtype 2 (HIV-2). Related viruses that are used as animal models includesimian immunodeficiency virus (SIV), and feline immunodeficiency virus(FIV). Treatment of HIV-1 with HAART has been effective in reducing theviral burden and ameliorating the effects of HIV-1 infection in infectedindividuals.

HXB2 numbering system: A reference numbering system for HIV protein andnucleic acid sequences, using HIV-1 HXB2 strain sequences as a referencefor all other HIV strain sequences. The person of ordinary skill in theart is familiar with the HXB2 numbering system, and this system is setforth in “Numbering Positions in HIV Relative to HXB2CG,” Bette Korberet al., Human Retroviruses and AIDS 1998: A Compilation and Analysis ofNucleic Acid and Amino Acid Sequences. Korber B, Kuiken C L, Foley B,Hahn B, McCutchan F, Mellors J W, and Sodroski J, Eds. TheoreticalBiology and Biophysics Group, Los Alamos National Laboratory, LosAlamos, N. Mex., which is incorporated by reference herein in itsentirety. HXB2 is also known as: HXBc2, for HXB clone 2; HXB2R, in theLos Alamos HIV database, with the R for revised, as it was slightlyrevised relative to the original HXB2 sequence; and HXB2CG in GENBANK™,for HXB2 complete genome. The numbering used in gp41 polypeptidesdisclosed herein is relative to the HXB2 numbering scheme.

IgA: A polypeptide belonging to the class of antibodies that aresubstantially encoded by a recognized immunoglobulin alpha gene. Inhumans, this class or isotype includes IgA₁ and IgA₂. IgAn antibodiescan exist as monomers, polymers (referred to as pIgA) of predominantlydimeric form, and secretory IgA. The constant chain of wild-type IgAcontains an 18-amino-acid extension at its C-terminus called the tailpiece (tp). Polymeric IgA is secreted by plasma cells with a 15-kDapeptide called the J chain linking two monomers of IgA through theconserved cysteine residue in the tail piece.

IgG: A polypeptide belonging to the class or isotype of antibodies thatare substantially encoded by a recognized immunoglobulin gamma gene. Inhumans, this class includes IgG₁, IgG₂, IgG₃, and IgG₄.

Immune complex: The binding of antibody to a soluble antigen forms animmune complex. The formation of an immune complex can be detectedthrough conventional methods known to the skilled artisan, for instanceimmunohistochemistry, immunoprecipitation, flow cytometry,immunofluorescence microscopy, ELISA, immunoblotting (for example,Western blot), magnetic resonance imaging, CT scans, X-ray and affinitychromatography. Immunological binding properties of selected antibodiesmay be quantified using methods well known in the art.

Immunoadhesin: A molecular fusion of a protein with the Fc region of animmunoglobulin, wherein the immunoglobulin retains specific properties,such as Fc receptor binding and increased half-life. An Fc fusioncombines the Fc region of an immunoglobulin with a fusion partner, whichin general can be any protein, polypeptide, peptide, or small molecule.In one example, an immunoadhesin includes the hinge, CH₂, and CH₃domains of the immunoglobulin gamma 1 heavy chain constant region. Inanother example, the immunoadhesin includes the CH₂, and CH₃ domains ofan IgG.

Immunogen: A compound, composition, or substance (for example, a proteinor a portion thereof) that is capable of inducing an immune response ina mammal, such as a mammal infected or at risk of infection with apathogen. Administration of an immunogen can lead to protective immunityand/or proactive immunity against a pathogen of interest. In someexamples, an immunogen is an HIV antigen. Examples of immunogensinclude, but are not limited to, peptides, lipids, polysaccharides,combinations thereof, and nucleic acids containing antigenicdeterminants, such as those recognized by an immune cell. In someexamples, immunogens include peptides derived from a pathogen ofinterest. Exemplary pathogens include bacteria, fungi, viruses andparasites. In specific examples, an immunogen is derived from HIV, suchas a gp41 polypeptide derived from HIV or antigenic fragment thereof.

Immunological Probe: A molecule that can be used for selection ofantibodies from sera which are directed against a specific epitope,including from human patient sera. The epitope scaffolds, along withrelated point mutants, can be used as immunological probes in bothpositive and negative selection of antibodies against the epitope graft.In some examples immunological probes are engineered variants of gp120.

Immunologically reactive conditions: Includes reference to conditionswhich allow an antibody raised against a particular epitope to bind tothat epitope to a detectably greater degree than, and/or to thesubstantial exclusion of, binding to substantially all other epitopes.Immunologically reactive conditions are dependent upon the format of theantibody binding reaction and typically are those utilized inimmunoassay protocols or those conditions encountered in vivo. SeeHarlow & Lane, infra, for a description of immunoassay formats andconditions. The immunologically reactive conditions employed in themethods are “physiological conditions” which include reference toconditions (e.g., temperature, osmolarity, and pH) that are typicalinside a living mammal or a mammalian cell. While it is recognized thatsome organs are subject to extreme conditions, the intra-organismal andintracellular environment normally lies around pH 7 (e.g., from pH 6.0to pH 8.0, more typically pH 6.5 to 7.5), contains water as thepredominant solvent, and exists at a temperature above 0° C. and below50° C. Osmolarity is within the range that is supportive of cellviability and proliferation.

Inhibiting or treating a disease: Inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease such as acquired immunodeficiency syndrome (AIDS). “Treatment”refers to a therapeutic intervention that ameliorates a sign or symptomof a disease or pathological condition after it has begun to develop.The term “ameliorating,” with reference to a disease or pathologicalcondition, refers to any observable beneficial effect of the treatment.The beneficial effect can be evidenced, for example, by a delayed onsetof clinical symptoms of the disease in a susceptible subject, areduction in severity of some or all clinical symptoms of the disease, aslower progression of the disease, a reduction in the viral load, animprovement in the overall health or well-being of the subject, or byother parameters well known in the art that are specific to theparticular disease. A “prophylactic” treatment is a treatmentadministered to a subject who does not exhibit signs of a disease orexhibits only early signs for the purpose of decreasing the risk ofdeveloping pathology.

Interleukin-2 (IL-2): IL-2 is a cytokine that is necessary for thegrowth and function of T cells. Antigen binding to the T cell receptor(TCR) stimulates the secretion of IL-2, and the expression of IL-2receptors IL-2R. The IL-2/IL-2R interaction then stimulates the growth,differentiation and survival of antigen-selected cytotoxic T cells viathe activation of the expression of specific genes. As such, IL-2 isnecessary for the development of T cell immunologic memory, whichdepends upon the expansion of the number and function ofantigen-selected T cell clones. IL-2 is also necessary during T celldevelopment in the thymus for the maturation of a subset of T cells thatare termed regulatory T cells. An exemplary amino acid sequence of humanIL-2 is provided in GENBANK® Accession No. NM_000586 (Jun. 10, 2012),which is incorporated by reference herein.

Interleukin-21 (IL-21): A cytokine cloned from a cDNA library derivedfrom activated CD3+ T cells (Parrish-Novak et al., Nature 408:57-63,2000). The IL-21 cDNA encodes a secreted protein of 131 amino acidsprotein most closely related to IL-2 and IL-15. The IL-21 gene has beenmapped to human chromosome 4q26-q27 near the IL-2 gene.

IL-21 mRNA has been demonstrated to be expressed in activated CD4+cells,but not in other T cells, B cells, or monocytes (Parrish-Novak et al.,Nature 408:57-63, 2000). However, it has been demonstrated that IL-21stimulates proliferation of B cells that are stimulated by cross-linkingof the CD40 antigen and proliferation of B cells stimulated by IL-4 inaddition to anti-IgM. IL-21 has also been shown to stimulateproliferation of naive (CD45RA (+)) cells, mediated by engagement ofCD3. IL-21 has also been shown to stimulate the proliferation of bonemarrow progenitor to cells and to enhance the expression of the NK cellmarker CD56 in the presence of IL-15. (For review, see Horst Ibelgaufts'COPE: Cytokines Online Pathfinder Encyclopedia, available on theinternet). The amino acid sequence of an exemplary human IL-21 is shownas SEQ ID NO: 1 in U.S. Published Patent Application No. 2003/0003545,which is incorporated herein, by reference. A representative clonecontaining all or most of the sequence for IL-21 (designated HTGED19)was deposited with the American Type Culture Collection (“ATCC”) on Mar.5, 1998, and was given the ATCC Deposit Number 209666 (see, e.g., U.S.Published Patent Application No. 2003/0003545).

The IL-21 receptor has been isolated and was found to be expressed byCD23+ B cells, B cell lines, a T cell leukemia line, and NK cell lines.The receptor gene has been mapped to human chromosome 16p12 (seeParrish-Novak et al., Nature 408:57-63, 2000; Ozaki et al., Proc. Natl.Acad. Sci. USA 97:11439-11444, 2000).

Interleukin 15 (IL-15): A cytokine with structural similarity to IL-2.IL-15 binds to and signals through the IL-2/IL-15 beta chain (CD122) andthe common gamma chain (gamma-C, CD132). IL-15 is secreted bymononuclear phagocytes (and some other cells) following infection byvirus(es). This cytokine induces cell proliferation of natural killercells; cells of the innate immune system whose principal role is to killvirally infected cells.

Isolated: An “isolated” biological component (such as an antibody, forexample, an antibody that specifically binds gp41, a nucleic acid,peptide, protein or antibody) has been substantially separated, producedapart from, or purified away from other biological components in thecell of the organism in which the component naturally occurs, such as,other chromosomal and extrachromosomal DNA and RNA, and proteins.Nucleic acids, peptides and proteins which have been “isolated” thusinclude nucleic acids and proteins purified by standard purificationmethods. The term also embraces nucleic acids, peptides, and proteinsprepared by recombinant expression in a host cell as well as chemicallysynthesized nucleic acids or polypeptides. In some examples, anantibody, such as an antibody specific for gp41, can be isolated, forexample, isolated from a subject infected with HIV.

An “isolated” cell is a cell that has been purified from the othercellular components of a tissue. Cells can be isolated by mechanical(such as by use FACS) and/or enzymatic methods. In several embodiments,an isolated population of cells (such as a B cell repertoire) includesgreater than about 80%, about 85%, about 90%, about 95%, or greater thanabout 99% of the cells of interest. In another embodiment, an isolatedpopulation of cells is one in which no other cells of a differentphenotype can be detected. In a further embodiment, an isolatedpopulation of cells is a population of cells that includes less thanabout 20%, about 15%, about 10%, about 5%, or less than about 1% of acells of a different phenotype than the cells of interest.

K_(D): The dissociation constant for a given interaction, such as apolypeptide ligand interaction or an antibody antigen interaction. Forexample, for the bimolecular interaction of an antibody (such as onedisclosed herein) and an antigen (such as gp41) it is the concentrationof the individual components of the bimolecular interaction divided bythe concentration of the complex.

Linker: A bi-functional molecule that can be used to link two moleculesinto one contiguous molecule, for example, to link an effector moleculeto an antibody. In some embodiments, a conjugate includes a linkerbetween the effector molecule or detectable marker and an antibody. Insome embodiments, the linker is cleavable under intracellularconditions, such that cleavage of the linker releases the effectormolecule or detectable marker from the antibody in the intracellularenvironment. In yet other embodiments, the linker is not cleavable andthe effector molecule or detectable marker can be released, for example,by antibody degradation. In some cases, a linker is a peptide within anantibody binding fragment (such as an Fv fragment) which serves toindirectly bond the variable heavy chain to the variable light chain.

The terms “conjugating,” “joining,” “bonding” or “linking” refer tomaking two polypeptides into one contiguous polypeptide molecule, tocovalently attaching a radionuclide or other molecule to a polypeptide,such as an antibody that specifically binds gp41, or an antibody bindingfragment thereof. In the specific context, the terms include referenceto joining a ligand, such as an antibody moiety, to an effectormolecule. The linkage can be either by chemical or recombinant means.“Chemical means” refers to a reaction between the antibody moiety andthe effector molecule such that there is a covalent bond formed betweenthe two molecules to form one molecule.

Membrane-proximal external region (MPER) of gp41: A region that isimmediately N-terminal of the transmembrane region of gp41. The MPER ishighly hydrophobic (50% of residues are hydrophobic) and is highlyconserved across many HIV clades (Zwick, M. B., et al., J Virol, 75(22): p. 10892-905, 2001). The conserved MPER of HIV-1 gp41 is a targetof two neutralizing human monoclonal antibodies, 2F5 and 4E10. The coreof the 2F5 epitope has been shown to be ELDKWAS (SEQ ID NO: 9). Withthis epitope, the residues D, K, and W were found to be most criticalfor recognition by 2F5. The core of the 4E10 epitope, NWFDIT (SEQ ID NO:10), maps just C-terminal to the 2F5 epitope on the gp41 ectodomain.

Neutralizing antibody: An antibody which reduces the infectious titer ofan infectious agent by binding to a specific antigen on the infectiousagent. In some examples the infectious agent is a virus. In someexamples, an antibody that is specific for gp41 neutralizes theinfectious titer of HIV. A “broadly neutralizing antibody” is anantibody that binds to and inhibits the function of related antigens,such as antigens that share at least 85%, 90%, 95%, 96%, 97%, 98% or 99%identity antigenic surface of antigen. With regard to an antigen from apathogen, such as a virus, the antibody can bind to and inhibit thefunction of an antigen from more than one class and/or subclass of thepathogen. For example, with regard to a human immunodeficiency virus,the antibody can bind to and inhibit the function of an antigen, such asgp41 from more than one clade. In one embodiment, broadly neutralizingantibodies to HIV are distinct from other antibodies to HIV in that theyneutralize a high percentage of the many types of HIV in circulation.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Recombinant nucleic acid” refers to a nucleic acid having nucleotidesequences that are not naturally joined together. This includes nucleicacid vectors including an amplified or assembled nucleic acid which canbe used to transform a suitable host cell. A host cell that includes therecombinant nucleic acid is referred to as a “recombinant host cell.”The gene is then expressed in the recombinant host cell to produce,e.g., a “recombinant polypeptide.” A recombinant nucleic acid may servea non-coding function (e.g., promoter, origin of replication,ribosome-binding site, etc.) as well.

A first sequence is an “antisense” with respect to a second sequence ifa polynucleotide whose sequence is the first sequence specificallyhybridizes with a polynucleotide whose sequence is the second sequence.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter, such as the CMV promoter, isoperably linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequence. Generally, operablylinked DNA sequences are contiguous and, where necessary to join twoprotein-coding regions, in the same reading frame.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell. In some examples a pharmaceuticalagent includes one or more of the disclosed antibodies.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995,describes compositions and formulations suitable for pharmaceuticaldelivery of the antibodies herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually include injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). In one embodiment, the polypeptide is gp41polypeptide. In one embodiment, the polypeptide is a disclosed antibodyor a fragment thereof. A “residue” refers to an amino acid or amino acidmimetic incorporated in a polypeptide by an amide bond or amide bondmimetic. A polypeptide has an amino terminal (N-terminal) end and acarboxy terminal (C-terminal) end.

Promoter: A promoter is an array of nucleic acid control sequences thatdirects transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription, forexample, in the case of a polymerase II type promoter, a TATA element. Apromoter also optionally includes distal enhancer or repressor elementswhich can be located as much as several thousand base pairs from thestart site of transcription. Both constitutive and inducible promotersare included (see for example, Bitter et al., Methods in Enzymology153:516-544, 1987).

Specific, non-limiting examples of promoters include promoters derivedfrom the genome of mammalian cells (such as the metallothioneinpromoter) or from mammalian viruses (such as the retrovirus longterminal repeat; the adenovirus late promoter; the vaccinia virus 7.5Kpromoter) may be used. Promoters produced by recombinant DNA orsynthetic techniques may also be used. A polynucleotide can be insertedinto an expression vector that contains a promoter sequence whichfacilitates the efficient transcription of the inserted genetic sequenceof the host. The expression vector typically contains an origin ofreplication, a promoter, as well as specific nucleic acid sequences thatallow phenotypic selection of the transformed cells.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein (such as an antibody)is more enriched than the peptide or protein is in its naturalenvironment within a cell. In one embodiment, a preparation is purifiedsuch that the protein or peptide represents at least 50% of the totalpeptide or protein content of the preparation.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a polypeptide will possess a relatively highdegree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a V_(L) or a V_(H) of an antibody thatspecifically binds a polypeptide are typically characterized bypossession of at least about 75%, for example at least about 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identitycounted over the full length alignment with the amino acid sequence ofinterest. Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% sequence identity. When less than theentire sequence is being compared for sequence identity, homologs andvariants will typically possess at least 80% sequence identity overshort windows of 10-20 amino acids, and may possess sequence identitiesof at least 85% or at least 90% or 95% depending on their similarity tothe reference sequence. Methods for determining sequence identity oversuch short windows are available at the NCBI website on the internet.One of skill in the art will appreciate that these sequence identityranges are provided for guidance only; it is entirely possible thatstrongly significant homologs could be obtained that fall outside of theranges provided.

Specifically bind: When referring to an antibody, refers to a bindingreaction which determines the presence of a target protein, peptide, orpolysaccharide in the presence of a heterogeneous population of proteinsand other biologics. Thus, under designated conditions, an antibodybinds preferentially to a particular target protein, peptide orpolysaccharide (such as an antigen present on the surface of a pathogen,for example gp41) and do not bind in a significant amount to otherproteins or polysaccharides present in the sample or subject. Specificbinding can be determined by methods known in the art, such as bymeasuring the affinity of the antibody for an antigen. In oneembodiment, affinity is calculated by a modification of the Scatchardmethod described by Frankel et al., Mol. Immunol., 16:101-106, 1979. Inanother embodiment, binding affinity is measured by an antigen/antibodydissociation rate. In yet another embodiment, a high binding affinity ismeasured by a competition radioimmunoassay. With reference to anantibody antigen complex, specific binding of the antigen and antibodyhas a K_(d) of less than about 10⁻⁶ Molar, such as less than about 10⁻⁶Molar, 10⁻⁷ Molar, 10⁻⁸ Molar, 10⁻⁹, or even less than about 10⁻¹⁰Molar.

Substantially purified: The term substantially purified indicates thatthe subject is substantially free of other molecular or cellularconstituents with which it is naturally associated. Thus, asubstantially purified population of cells (such as B cells, B cellprogenitors, mature B cells, memory B cells, plasma cells, etc.) issubstantially free of other cellular components of the tissue in whichit is naturally found, such as bone marrow, peripheral blood, spleen,lymph node, etc. For example, a substantially pure population of B cells(e.g., a B cell progenitor, an immature B cell, a mature B cell, amemory B cell, a plasma cell, etc.) is at least 50%, for example atleast about 80% or alternatively at least about 90% free of othercellular components. In an embodiment, the population of B cells is atleast about 95% free of other cells. For example, a population ofpurified B cells, obtained from a tissue such as peripheral blood, issubstantially free of red blood cells, T cells, platelets, and othercells typically found in peripheral blood.

T Cell: A white blood cell critical to the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4). These cells, also known ashelper T cells, help orchestrate the immune response, including antibodyresponses as well as killer T cell responses. CD8⁺ T cells carry the“cluster of differentiation 8” (CD8) marker. In one embodiment, a CD8 Tcells is a cytotoxic T lymphocytes. In another embodiment, a CD8 cell isa suppressor T cell.

Therapeutic agent: Used in a generic sense, it includes treating agents,prophylactic agents, and replacement agents. A therapeutic agent is usedto ameliorate a specific set of conditions in a subject with a diseaseor a disorder.

Therapeutically effective amount or effective amount: A quantity of aspecific substance, such as a disclosed antibody, sufficient to achievea desired effect in a subject being treated. For instance, this can bethe amount necessary to inhibit HIV replication or treat AIDS. Inseveral embodiments, a therapeutically effective amount is the amountnecessary to reduce a sign or symptom of AIDS, and/or to decrease viraltiter in a subject. When administered to a subject, a dosage willgenerally be used that will achieve target tissue concentrations thathas been shown to achieve a desired in vitro effect.

Toxin: An effector molecule that induces cytotoxicity when it contacts acell. Specific, non-limiting examples of toxins include, but are notlimited to, abrin, ricin, auristatins (such as monomethyl auristatin E(MMAE; see for example, Francisco et al., Blood, 102: 1458-1465, 2003))and monomethyl auristatin F (MMAF; see, for example, Doronina et al.,BioConjugate Chem., 17: 114-124, 2006), maytansinoids (such as DM1; see,for example, Phillips et al., Cancer Res., 68:9280-9290, 2008),Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40),diphtheria toxin (DT), botulinum toxin, saporin, restrictocin orgelonin, or modified toxins thereof, or other toxic agents that directlyor indirectly inhibit cell growth or kill cells. For example, PE and DTare highly toxic compounds that typically bring about death throughliver toxicity. PE and DT, however, can be modified into a form for useas an immunotoxin by removing the native targeting component of thetoxin (such as the domain Ia of PE and the B chain of DT) and replacingit with a different targeting moiety, such as an antibody.

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits a desired activity. In one example the desiredactivity is formation of an immune complex. In particular examples thedesired activity is treatment of a tumor.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Virus: Microscopic infectious organism that reproduces inside livingcells. A virus consists essentially of a core of a single nucleic acidsurrounded by a protein coat, and has the ability to replicate onlyinside a living cell. “Viral replication” is the production ofadditional virus by the occurrence of at least one viral life cycle. Avirus may subvert the host cells' normal functions, causing the cell tobehave in a manner determined by the virus. For example, a viralinfection may result in a cell producing a cytokine, or responding to acytokine, when the uninfected cell does not normally do so.

“Retroviruses” are RNA viruses wherein the viral genome is RNA. When ahost cell is infected with a retrovirus, the genomic RNA is reversetranscribed into a DNA intermediate which is integrated very efficientlyinto the chromosomal DNA of infected cells. The integrated DNAintermediate is referred to as a provirus. The term “lentivirus” is usedin its conventional sense to describe a genus of viruses containingreverse transcriptase. The lentiviruses include the “immunodeficiencyviruses” which include human immunodeficiency virus (HIV) type 1 andtype 2 (HIV-I and HIV-II), simian immunodeficiency virus (SIV), andfeline immunodeficiency virus (FIV).

Suitable methods and materials for the practice or testing of thisdisclosure are described below. Such methods and materials areillustrative only and are not intended to be limiting. Other methods andmaterials similar or equivalent to those described herein can be used.For example, conventional methods well known in the art to which adisclosed invention pertains are described in various general and morespecific references, including, for example, Sambrook et al., MolecularCloning: A Laboratory Manual, 2d ed., Cold Spring Harbor LaboratoryPress, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3ded., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocolsin Molecular Biology, Greene Publishing Associates, 1992 (andsupplements to 2012); Ausubel et al., Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlowand Lane, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, 1999.

II. Description of Several Embodiments

A. Neutralizing Monoclonal Antibodies

Isolated human monoclonal antibodies that specifically bind gp41 aredisclosed herein. The disclosed antibodies specifically bind themembrane-proximal extracellular region (MPER) of gp41. Also disclosedherein are compositions including these human monoclonal antibodies anda pharmaceutically acceptable carrier. Nucleic acids encoding theseantibodies, expression vectors including these nucleic acids, andisolated host cells that express the nucleic acids are also provided.

Compositions including the human monoclonal antibodies specific for gp41can be used for research, diagnostic and therapeutic purposes. Forexample, the human monoclonal antibodies disclosed herein can be used todetect HIV-1 in a biological sample or interfere with the HIV-1activity, for example to diagnose or treat a subject having an HIV-1infection and/or AIDS. For example, the antibodies can be used todetermine HIV-1 titer in a subject. The antibodies disclosed herein alsocan be used to study the biology of the human immunodeficiency virus.

The disclosed antibodies that specifically bind gp41 bind the membraneproximal extracellular region (MPER) of gp41 at a previouslyuncharacterized epitope, that is designated herein as the 10E8 epitope,for the first member of this class of antibodies discovered (10E8-likeantibodies). The crystal structure of the 10E8 antibody was solved incomplex with a gp41 peptide (see Example 1), which allowed for detailedanalysis of the binding of this class of the 10E8 antibody and gp41, anddescribe at the atomic level the binding of 10E8-like antibodies (suchas 10E8) to the 10E8 epitope. This epitope, and thus the antibodies ofthis class (10E8-like antibodies), can be distinguished from otherantibodies that bond gp41 by virtue of their binding to the 10E8epitope. In several embodiments, the 10E8 epitope, e.g.,KWASLWNWFDITNWLWYIR (SEQ ID NO: 13), extends C-terminal to the 2F5epitope (although there is some overlap) on the gp41 ectodomain and isdistinguished from the 4E10 and Z13E1 epitope by expanding the bindingto C-terminal residues previously thought to be inaccessible (e.g. theseresidues were believed to be buried in the lipid bilayer). The person ofordinary skill in the art will understand that the 10E8 antibodies canspecifically bind to gp41MPER residues extending N-terminal to the abovesequence. In some embodiments, the disclosed 10E8-like antibodiesspecifically bind to a polypeptide including an amino acid sequence setforth as residues 1-28, 2-28, 3-28, 4-28, 5-28, 6-28, 7-28, 8-28, 9-28,10-28, 11-28, 12-28, 13-28 or 14-28 of SEQ ID NO: 26, which correspondto gp41 residues 656-683, 657-683, 658-683, 659-683, 660-683, 661-683,662-683, 663-683, 664-683, 665-683, 666-683, 667-683, 668-683, or669-683, respectively (HXB2 numbering system).

In some embodiments, residues believed to make contacts with the 10E8antibody include the K, SLWNWF, TN, LW, and IR shown in bold above.Thus, in some embodiments a 10E8-like antibody specifically binds to oneor more of K, SLWNWF, TN, LW, and IR of SEQ ID NO: 13, such as at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, at least 10, at least 11, at least 12 or evenall 13 of these residues. In some examples, a 10E8-like antibody bindsto the NWF, T and R resides shown in bold in the following sequenceNWFDITNWLWYIR (residues 7-19 of SEQ ID NO: 13).

In additional embodiments, the antibody or antigen binding fragmentcontacts L, WF, LW and R shown in bold in the amino acid sequence setforth as LWNWFDITNWLWYIR (SEQ ID NO: 26, residues 14-28). In additionalembodiments, the disclosed antibody contacts LW, WF, LW and R shown inbold in the amino acid sequence set forth as LWNWFDITNWLWYIR (SEQ ID NO:26, residues 14-28). In additional embodiments, the disclosed antibodycontacts SLW, WF, LW and R shown in bold in the amino acid sequence setforth as SLWNWFDITNWLWYIR (SEQ ID NO: 26, residues 13-28). In additionalembodiments, the disclosed antibody contacts L, DK, SLWNWF, TN, LW andIR shown in bold in the amino acid sequence set forth asLELDKWASLWNWFDITNWLWYIR (SEQ ID NO: 26, residues 6-28). In additionalembodiments, the disclosed antibody contacts NWF, T, and R shown in boldin the amino acid sequence set forth as NWFDITNWLWYIR (SEQ ID NO: 13,residues 7-19). In additional embodiments, the disclosed antibodycontacts K, SLNWF, T, and IR shown in bold in the amino acid sequenceset forth as KWASLWNWFDITNWLWYIR (SEQ ID NO: 13). In additionalembodiments, the disclosed antibody the antibody specifically binds toresidues NWF, T, and R shown in bold in the amino acid sequence setforth as NWFDITNWLWYIR (SEQ ID NO: 13, residues 7-19). In additionalembodiments, the disclosed antibody specifically binds to residues K,SLNWF, T, and IR shown in bold in the amino acid sequence set forth asKWASLWNWFDITNWLWYIR (SEQ ID NO: 13). In several such embodiments, theantibody directly contacts the gp41MPER at the indicated residues whenspecifically bound to gp41, for example, through hydrogen bond contactsand/or Van der Waals contacts. In additional embodiments, the antibodycontacts the gp41MPER at the indicated residues when specifically boundto gp41, for example through hydrogen bond contacts, Van der Waalscontacts, and/or interactions that cause reduced solvent access betweenthe antibody and gp41 (i.e., buried surface area). As shown in FIGS. 27and 28, residues in the 10E8 and 10E8-like antibodies that are importantfor binding to the 10E8 epitope include Kabat residues 28, 31, 33, 50,52, 52B, 52C, 53, 56, 58, and 97-100J of the heavy chain and Kabatresidues 91 and 95B of the light chain. These residues correspond toresidues 28, 31, 33, 50, 52, 54, 55, 56, 59, 61, 103-116 of the heavychain (the residue numbers are given relative to SEQ ID NO: 1), and 91and 97 of the light chain (the residue numbers are given relative to SEQID NO: 2). In some embodiments, a 10E8-like antibody specifically bindsto gp41, and one or more of residues 28, 31, 33, 50, 52, 54, 55, 56, 59,61, and/or 103-116 from the heavy chain (relative to SEQ ID NO: 1), suchas at least 1, at least 2, at least 3, at least 4, at least 5, at least6, at least 7, at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, or even all 27 of theseresidues contact gp41. In some embodiments, a 10E8-like antibodyspecifically binds to gp41 and at least one of residues 91 and 97 fromthe light chain (relative to SEQ ID NO: 2) contact gp41.

In some embodiments, the class of 10E8-like antibodies do not exhibitself-reactivity, that is they do not bind self-antigens, such as humanprotein. Without being bound by theory, examination of the crystalstructure of 10E8 in complex with an MPER gp41 peptide shows that 10E8binds to the MPER in a manner that may not require any hydrophobicinteraction with membrane. Other known neutralizing antibodies that bindthe MPER of gp41, such as 2F5 and 4E10, include hydrophobic residues inthe CDR H3 that do not contact the epitope and are believed to makespecific contacts with the lipid membrane in which gp41 is situated.

While not being bound by theory, it is believed that the neutralizationbreadth of the 10E8-like antibodies can tolerate conservative changes tothe epitope while still maintaining binding. For example, while theC-terminal residue is shown as an arginine, antibodies of this class cantolerate a lysine substitution at this site, and still maintain highbinding affinity. In addition, one of ordinary skill in the art canformulate a consensus sequence for the 10E8 epitope using the sequencesof all of HIV gp41 variations for the HIV isolates listed in FIG. 17B orFIGS. 17C-17F. In some embodiments, the antibodies in this class(10E8-like antibodies) can also be distinguished by neutralizationbreadth. In some embodiments, a 10E8-like antibody can neutralize atleast 95% (such as at least 96%, at least 97%, at least 98% or at least99%) of the HIV-1 isolates listed in FIG. 17B or FIGS. 17C-17F with anIC50 of less than 50 μg/ml. In some embodiments, a 10E8-like antibodycan neutralize at least 65% (such as at least 66%, at least 67%, atleast 68%, at least 69%, at least 70%, at least 71%, at least 72%, atleast 73%, at least 74%, at least 75%, or at least 80%) of the HIV-1isolates listed in FIG. 17B or FIGS. 17C-17F with an IC50 of less than 1μg/ml. In specific embodiments, a 10E8-like antibody is not the Z13E1,4E10 or 2F5 antibody.

The discussion of monoclonal antibodies below refers to isolatedmonoclonal antibodies that include heavy and light chain variabledomains including a CDR1, CDR2 and CDR3. The person of ordinary skill inthe art will understand that various CDR numbering schemes (such as theKabat, Chothia or IMGT numbering schemes) can be used to determine CDRpositions. The heavy chain CDR positions of the 10E8 monoclonal antibodyaccording to the Kabat and IMGT numbering scheme are shown in FIG. 6A(Kabat) and FIG. 6B (IMGT). In several embodiments, reference toparticular amino acid substitutions in the heavy or light chains of thedisclosed antibodies is made according to the Kabat or IMGT numberingschemes. For example, the amino acid substitution S74W in 10E8referenced herein refers to the Kabat numbering scheme. Using the IMGTnumbering scheme this substitution would be referred to as S82W. In bothcases, this substation refers to substitution of the serine residue atposition 77 of SEQ ID NO: 1. The person of skill in the art will readilyunderstand use of various CDR numbering schemes when referencingparticular amino acids of the antibodies disclosed herein.

In some embodiments, an isolated antibody that specifically binds gp41includes a heavy chain with one or more of amino acids 26-33(complementarity-determining region 1 (CDR1)), amino acids 51-60 (CDR2),and/or 99-120 (CDR3) of SEQ ID NO: 11:

EVX₁LX₂ESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAAPVEGRFTISRLNX₃INFLYLEMNNLRMEDSGLYFCARTGKYYDFWSGYPPGEEYFQDWGRGTLVX₄VSS(SEQ ID NO: 11), where X₁ is Q or R, X₂ is V or A, X₃ is S or Y, and X₄is T or I. In some embodiments, an isolated human monoclonal antibodythat specifically binds gp41 includes a heavy chain with amino acids26-33 (CDR1), 51-60 (CDR2), and 99-120 (CDR3) of SEQ ID NO: 11. Inspecific examples, the heavy chain of the human monoclonal antibodyincludes SEQ ID NO: 11.

In some embodiments, an isolated antibody that specifically binds gp41includes a heavy chain with one or more of amino acids 26-33(complementarity-determining region 1 (CDR1)), amino acids 51-60 (CDR2),and/or 99-120 (CDR3) of SEQ ID NO: 146:

EVX₁LX₂ESGGGLVKPGGSLRLSCSASGFX₃FX₄X₅AWMTWVRQPPGKGLEWVGRITGPGEX₆WSVDYAAPVEGRFTISRLNSINFLYLEMNNLRMEDSGLYFCARTGKYYDFWSGYPPGEEYFQDWGRGTLVX₇VSS (SEQ ID NO: 146), where X₁ is Q or R, X₂ is V or A, X₃ is Dor W, X₄ is D or W, X₅ is N or W, X₆ is G or W and X₇ is T or I). Insome embodiments, an isolated human monoclonal antibody thatspecifically binds gp41 includes a heavy chain with amino acids 26-33(CDR1), 51-60 (CDR2), and 99-120 (CDR3) of SEQ ID NO: 146. In specificexamples, the heavy chain of the human monoclonal antibody includes SEQID NO: 146.

In some embodiments, an isolated antibody that specifically binds gp41includes a heavy chain with one or more of amino acids 26-33(complementarity-determining region 1 (CDR1)), amino acids 51-60 (CDR2),and/or 99-120 (CDR3) of SEQ ID NO: 187. SEQ ID NO: 187 is set forth asEX₁X₂LX₃ESGGX₄LVX₅PGGSLRLSCX₆ASGFX₇FX₈X₉X₁₀WMTWVRQX₁₁PGKGLEWVGRIX₁₂-GX₁₃GX₁₄X₁₅WX₁₆X₁₇X₁₈YAX₁₉X₂₀VX₂₁GRFX₂₂ISRX₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉YLX₃₀MNX₃₁X₃₂X₃₃X₃₄X₃₅DX₃₆X₃₇X₃₈YX₃₉CX₄₀X₄₁TX₄₂KX₄₃YX₄₄FWX₄₅GX₄₆PPGEEYX₄₇X₄₈X₄₉WGX₅₀GTX₅₁VX₅₂VX₅₃S,wherein X₁ is V or I; X₂ is Q or R; X₃ is V or A; X₄ is G, R, or D; X₅is K or R; X₆ is S or A; X₇ is D, N, S, A, or W; X₈ is D, K, W or A; X₉is N, S, D, A, W, F, or Y; X₁₀ is A, T, or Q; X₁₁ is P, or A; X₁₂ is T,S, or A; X₁₃ is P or W; X₁₄ is E, A, F, L, M, V, or W; X₁₅ is G or W;X₁₆ is S, T, A, or H; X₁₇ is V or S; X₁₈ is D, G, or A; X₁₉ is A or E;X₂₀ is P, S, or T; X₂₁ is E, K or Q; X₂₂ is T or I; X₂₃ is L, D, M, I,or N; X₂₄ is N or D; X₂₅ is 5, M, W, F, L, or M; X₂₆ is I or K; X₂₇ is Nor D; X₂₈ is F, T. or M; X₂₉ is L or F; X₃₀ is E or Q; X₃₁ is N, S, orR; X₃₂ is L or V; X₃₃ is R, or K; X₃₄ is M, T, I, or P; X₃₅ is E or D;X₃₆ is S, T or W; X₃₇ is G, A, or W; X₃₈ is L, V, S or Y; X₃₉ is F, orY; X₄₀ is A, T or V; X₄₁ is R, T, or H; X₄₂ is G or E; X₄₃ is Y or H;X₄₄ is D, A, or N; X₄₅ is S, G, or R; X₄₆ is Y or A; X₄₇ is F or L; X₄₈is Q or E; X₄₉ is D or H; X₅₀ is R or Q; X₅₁ is L or Q; X₅₂ is T or I;and X₅₃ is S or P.) In some embodiments, an isolated human monoclonalantibody that specifically binds gp41 includes a heavy chain with aminoacids 26-33 (CDR1), 51-60 (CDR2), and 99-120 (CDR3) of SEQ ID NO: 187.In additional embodiments, an isolated antibody that specifically bindsgp41 includes a heavy chain variable region including the amino acidsequence set forth as SEQ ID NO: 187.

In several embodiments, the isolated antibody that specifically bindsgp41, is neutralizing, and includes a heavy chain including one or moreof the heavy chain complementarity determining regions (CDRs) of one ofthe heavy chain variable region sequences set forth as SEQ ID NOs: 1, 3,5, 11, 146, 147-149, 187, 189-192, and 200-204, according to the Kabat,IMGT, or Clothia numbering systems. In some embodiments, an isolatedantibody that specifically binds gp41 and is neutralizing, includes aheavy chain including the CDR1, CDR2, and CDR3 of one of the heavy chainvariable region sequences set forth as SEQ ID NOs: 1, 3, 5, 11, 146,147-149, 187, 189-192, or 200-204, according to the Kabat, IMGT, orClothia numbering systems.

Thus in some embodiments, an isolated antibody that specifically bindsgp41 includes one or more of amino acids 26-33 (CDR1), amino acids 51-60(CDR2), and/or 99-120 (CDR3) of one of SEQ ID NOs: 1, 3, 5, 11, 146,147-149, 187, 189-192, and 200-204. In some embodiments, an isolatedhuman monoclonal antibody that specifically binds gp41 includes a heavychain with amino acids 26-33 (CDR1), 51-60 (CDR2), and 99-120 (CDR3) ofone of SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204.In specific examples, the heavy chain of the human monoclonal antibodyincludes one of SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and200-204.

For example, in some embodiments, an isolated antibody that specificallybinds gp41 includes a heavy chain including one or more of the heavychain complementarity determining regions (CDRs) from gp41 antibody10E8, 7H6 and/or 7N16. The heavy chain of gp41 antibody 10E8 is setforth as SEQ ID NO: 1. In some embodiments, an isolated antibody thatspecifically binds gp41 includes one or more of amino acids 26-33 (27-38in FIG. 6B) (CDR1), amino acids 51-60 (56-65 in FIG. 6B) (CDR2), and/or99-120 (105-126 in FIG. 6B) (CDR3) of SEQ ID NO: 1. In some embodiments,an isolated human monoclonal antibody that specifically binds gp41includes a heavy chain with amino acids 26-33 (CDR1), 51-60 (CDR2), and99-120 (CDR3) of SEQ ID NO: 1. In specific examples, the heavy chain ofthe human monoclonal antibody includes SEQ ID NO: 1. The heavy chain ofgp41 antibody 7H6 is set forth as SEQ ID NO: 3. Thus, in someembodiments, an isolated antibody that specifically binds gp41 includesone or more of amino acids 26-33 (CDR1), 51-60 (CDR2), and/or 99-120(CDR3) of SEQ ID NO: 3. In some embodiments, an isolated humanmonoclonal antibody that specifically binds gp41 includes a heavy chainwith amino acids 26-33 (CDR1), 51-60 (CDR2), and 99-120 (CDR3) of SEQ IDNO: 3. In specific examples, the heavy chain of the human monoclonalantibody includes SEQ ID NO: 3. The heavy chain of gp41 antibody 7N16 isset forth as SEQ ID NO: 5. Thus in some embodiments, an isolatedantibody that specifically binds gp41 includes one or more of aminoacids 26-33 (CDR1), 51-60 (CDR2), and/or 99-120 (CDR3) of SEQ ID NO: 5.In some embodiments, an isolated human monoclonal antibody thatspecifically binds gp41 includes a heavy chain with amino acids 26-33(CDR1), 51-60 (CDR2), and 99-120 (CDR3) of SEQ ID NO: 5. In specificexamples, the heavy chain of the human monoclonal antibody includes SEQID NO: 5.

In additional embodiments, an isolated antibody that specifically bindsgp41 includes a heavy chain including the amino acid sequence of any oneof the 10E8-like heavy chains disclosed herein and further including anamino acid substitution at position 77 (position 74 according to Kabatnumbering), such as a S77Y substitution (S74Y according to Kabatnumbering). In additional embodiments, an isolated antibody thatspecifically binds gp41 includes a heavy chain including one or more ofthe heavy chain complementarity determining regions (CDRs) from any oneof the 10E8-like heavy chains disclosed herein and further including anamino acid substitution at position 77 (position 74 according to Kabatnumbering), such as a S77Y substitution (S74Y according to Kabatnumbering). In additional embodiments, an isolated antibody thatspecifically binds gp41 includes a heavy chain including one or more ofthe heavy chain complementarity determining regions (CDRs) from one ofgp41 antibodies gVRC-H2dN152 or gVRC-H2dN152 with an amino acidsubstitution at position 77 (position 74 according to Kabat numbering).In some examples the amino acid substitution is a serine to tyrosinesubstitution. The heavy chain of gp41 antibody gVRC-H2dN152 is set forthas SEQ ID NO: 154. Thus in some embodiments, an isolated antibody thatspecifically binds gp41 includes one or more of amino acids 26-33(CDR1), 51-60 (CDR2), and/or 99-120 (CDR3) of SEQ ID NO: 154. In someembodiments, an isolated human monoclonal antibody that specificallybinds gp41 includes a heavy chain with amino acids 26-33 (CDR1), 51-60(CDR2), and 99-120 (CDR3) of SEQ ID NO: 154. In specific examples, theheavy chain of the human monoclonal antibody includes SEQ ID NO: 154.The heavy chain of gp41 antibody gVRC-H2dN152 with serine to tyrosinesubstitution at position 77 (position 74 using Kabat numbering) is setforth as SEQ ID NO: 192. Thus in some embodiments, an isolated antibodythat specifically binds gp41 includes one or more of amino acids 26-33(CDR1), 51-60 (CDR2), and/or 99-120 (CDR3) of SEQ ID NO: 192. In someembodiments, an isolated human monoclonal antibody that specificallybinds gp41 includes a heavy chain with amino acids 26-33 (CDR1), 51-60(CDR2), and 99-120 (CDR3) of SEQ ID NO: 192. In specific examples, theheavy chain of the human monoclonal antibody includes SEQ ID NO: 192.

In some embodiments, an isolated antibody that specifically binds gp41includes one or more of the light chain complementarity determiningregions (CDRs) of amino acids 26-31 (CDR1), 49-51 (CDR2), and/or 88-99(CDR3) of SEQ ID NO: 12:SYELTQX₁TGVSVALGRTVVTITCRGDSLRSHX₂ASWYQKKPGQAPX₃LLFYGKNNRPSGX₄PDRFSGSASGNRASLTIX₅GAQAEDX₆AX₇YYCSSRDKSGSRLSVFGGGTKLX₈VL (SEQ ID NO:12), where X₁ is E or D, X₂ is Y or H, X₃ is V or I, X₄ is V or I, X₅ isS or T, X₆ is D or E, X₇ is E or D, and X₈ is T or I. In someembodiments, an isolated human monoclonal antibody that specificallybinds gp41 includes a light chain with amino acids 26-31 (CDR1), 49-51(CDR2), and 88-99 (CDR3) of SEQ ID NO: 12. In specific examples, thelight chain of the human monoclonal antibody includes SEQ ID NO: 12.

In some embodiments, an isolated antibody that specifically binds gp41includes one or more of the light chain complementarity determiningregions (CDRs) of amino acids 26-31 (CDR1), 49-51 (CDR2), and/or 88-99(CDR3) of SEQ ID NO: 188:X₁X₂X₃LTQX₄X₅X₆VSVAX₇X₈X₉TVX₁₀ITCX₁₁GDSLRX₁₂X₁₃YX₁₄X₁₅WYQX₁₆X₁₇X₁₈X₁₉QAPX₂₀-LX₂X₂₂YX₂₃X₂₄X₂₅X₂₆RPSX₂₇X₂₈X₂₉DRFSX₃₀X₃A₃₂SGNX₃₃ASLTIX₃₄GAX₃₅X₃₆X₃₇DX₃₈AX₃₉YYCSSRDKSGSRLX₄₀X₄₁FGX₄₂GTX₄₃X₄₄X₄₅X₄₆X₄₇, wherein X₁ is S or A; X₂is Y or S; X₃ is E or D; X₄ is E or D; X₅ is T or P; X₆ is G, A, or T;X₇ is L or F; X₈ is G, K, or E; X₉ is R, Q, or K; X₁₀ is T or R; X₁₁ isR or Q; X₁₂ is S, R, or N; X₁₃ is H or Y; X₁₄ is A, V, or T; X₁₅ is S orG; X₁₆ is K, E, or Q; X₁₇ is K or R; X₁₈ is P or T; X₁₉ is G or R; X₂₀is I, V, or K; X₂₁ is L or V; X₂₂ is F, V, or I; X₂₃ is G or P; X₂₄ is Kor R; X₂₅ is N, D, or H; X₂₆ is N or I; X₂₇ is G, or P; X₂₈ is V or I;X₂₉ is P, H, or S; X₃₀ is G or A; X₃₁ is S or F; X₃₂ is A, T, or S; X₃₃is R or T; X₃₄ is S, A, or T; X₃₅ is Q or E; X₃₆ is A or G; X₃₇ is E orD; X₃₈ is D, E, or I; X₃₉ is E or D; X₄₀ is S, V; X₄₁ is V, T; X₄₂ is G,R; X₄₃ is K or E; X₄₄ is L, V, or R; X₄₅ is T, S, or A; X₄₆ is V, T, orG; and X₄₇ is L, V, or P. In some embodiments, an isolated humanmonoclonal antibody that specifically binds gp41 includes a light chainwith amino acids 26-31 (CDR1), 49-51 (CDR2), and 88-99 (CDR3) of SEQ IDNO: 188. In additional embodiments, an isolated antibody thatspecifically binds gp41 includes a light chain variable region includingthe amino acid sequence set forth as SEQ ID NO: 188.

Several embodiments include an isolated antibody that specifically bindsgp41, is neutralizing, and includes a light chain including one or moreof the light chain complementarity determining regions (CDRs) of one ofthe light chain variable region sequences set forth as SEQ ID NOs: 2, 4,6, 12, 150-152, 164-186, 188, or 197-199, according to the Kabat, IMGT,or Clothia numbering systems. In some embodiments, an isolated antibodythat specifically binds gp41 and is neutralizing, includes a light chainincluding the CDR1, CDR2, and CDR3 of one of the light chain variableregion sequences set forth as SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186,188, or 197-199, according to the Kabat, IMGT, or Clothia numberingsystems. In additional embodiments, an isolated antibody thatspecifically binds gp41 includes one or more of amino acids 26-31 (27-38in FIG. 6B) (CDR1), 49-51 (56-65 in FIG. 6B) (CDR2), and/or 88-99(105-116 in FIG. 6B) (CDR3) of one of SEQ ID NOs: 2, 4, 6, 12, 150-152,164-186, 188, or 197-199. In further embodiments, an isolated humanmonoclonal antibody that specifically binds gp41 includes a light chainwith amino acids 26-31 (CDR1), 49-51 (CDR2), and 88-99 (CDR3) of one ofSEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199. In specificexamples, the light chain of the human monoclonal antibody includes oneof SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199.

For example, in some embodiments, an isolated antibody that specificallybinds gp41 includes one or more of the light chain complementaritydetermining regions (CDRs) from gp41 antibody 10E8, 7H6 and/or 7N16. Thelight chain of gp41 antibody 10E8 is set forth as SEQ ID NO: 2. Thus insome embodiments, an isolated antibody that specifically binds gp41includes one or more of amino acids 26-31 (27-38 in FIG. 6B) (CDR1),49-51 (56-65 in FIG. 6B) (CDR2), and/or 88-99 (105-116 in FIG. 6B)(CDR3) of SEQ ID NO: 2. In some embodiments, an isolated humanmonoclonal antibody that specifically binds gp41 includes a light chainwith amino acids 26-31 (CDR1), 49-51 (CDR2), and 88-99 (CDR3) of SEQ IDNO: 2. In specific examples, the light chain of the human monoclonalantibody includes SEQ ID NO: 2. The light chain of gp41 antibody 7H6 isset forth as SEQ ID NO: 4. Thus in some embodiments, an isolatedantibody that specifically binds gp41 includes one or more of aminoacids 26-31 (CDR1), 49-51 (CDR2), and/or 88-99 (CDR3) of SEQ ID NO: 4.In some embodiments, an isolated human monoclonal antibody thatspecifically binds gp41 includes a light chain with amino acids 26-31(CDR1), 49-51 (CDR2), and 88-99 (CDR3) of SEQ ID NO: 4. In specificexamples, the light chain of the human monoclonal antibody includes SEQID NO: 4. The light chain of gp41 antibody 7N16 is set forth as SEQ IDNO: 6. Thus in some embodiments, an isolated antibody that specificallybinds gp41 includes one or more of amino acids 26-31 (CDR1), 49-51(CDR2), and/or 88-99 (CDR3) of SEQ ID NO: 6. In some embodiments, anisolated human monoclonal antibody that specifically binds gp41 includesa heavy chain with amino acids 26-31 (CDR1), 49-51 (CDR2), and 88-99(CDR3) of SEQ ID NO: 6. In specific examples, the heavy chain of thehuman monoclonal antibody includes SEQ ID NO: 6.

In additional embodiments, an isolated antibody that specifically bindsgp41 includes a heavy chain including one or more of the heavy chaincomplementarity determining regions (CDRs) from gp41 antibody 10E8gL03.The light chain of gp41 antibody 10E8gH03 is set forth as SEQ ID NO:152. Thus in some embodiments, an isolated antibody that specificallybinds gp41 includes one or more of amino acids 26-31 (CDR1), 49-51(CDR2), and/or 88-99 (CDR3) of SEQ ID NO: 152. In some embodiments, anisolated human monoclonal antibody that specifically binds gp41 includesa heavy chain with amino acids 26-31 (CDR1), 49-51 (CDR2), and 88-99(CDR3) of SEQ ID NO: 152. In specific examples, the heavy chain of thehuman monoclonal antibody includes SEQ ID NO: 152.

Additional embodiments include an isolated antibody that specificallybinds gp41 and is neutralizing, and includes a heavy chain including oneor more of the heavy chain complementarity determining regions (CDRs) ofone of the heavy chain variable region sequences set forth as SEQ IDNOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204, according tothe Kabat, IMGT, or Clothia numbering systems, and one or more of thelight chain complementarity determining regions (CDRs) of one of thelight chain variable region sequences set forth as SEQ ID NOs: 2, 4, 6,12, 150-152, 164-186, 188, or 197-199, according to the Kabat, IMGT, orClothia numbering systems, respectively. Additional embodiments includean isolated antibody that specifically binds gp41 and is neutralizing,and includes a heavy chain including the heavy chain complementaritydetermining region 1 (HCRD1), HCRD2, and HCDR3 of one of the heavy chainvariable region sequences set forth as SEQ ID NOs: 1, 3, 5, 11, 146,147-149, 187, 189-192, and 200-204, according to the Kabat, IMGT, orClothia numbering systems, and the light chain complementaritydetermining region 1 (HCRD1), HCRD2, and HCDR3 of one of the light chainvariable region sequences set forth as SEQ ID NOs: 2, 4, 6, 12, 150-152,164-186, 188, or 197-199, according to the Kabat, IMGT, or Clothianumbering systems, respectively.

Thus in some embodiments, an isolated antibody that specifically bindsgp41 includes a heavy chain including amino acids 26-33 (27-38 in FIG.6B) (CDR1), amino acids 51-60 (56-65 in FIG. 6B) (CDR2), and/or 99-120(105-126 in FIG. 6B) (CDR3) of one of SEQ ID NOs: 1, 3, 5, 11, 146,147-149, 187, 189-192, and 200-204, and a light chain including aminoacids 26-31 (27-38 in FIG. 6B) (CDR1), 49-51 (56-65 in FIG. 6B) (CDR2),and/or 88-99 (105-116 in FIG. 6B) (CDR3) of one of SEQ ID NOs: 2, 4, 6,12, 150-152, 164-186, 188, or 197-199. In additional embodiments, anisolated antibody that specifically binds gp41 includes a heavy chainincluding amino acids 26-33 (27-38 in FIG. 6B) (CDR1), amino acids 51-60(56-65 in FIG. 6B) (CDR2), and 99-120 (105-126 in FIG. 6B) (CDR3) of oneof SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204, anda light chain including amino acids 26-31 (27-38 in FIG. 6B) (CDR1),49-51 (56-65 in FIG. 6B) (CDR2), and 88-99 (105-116 in FIG. 6B) (CDR3)of one of SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199.

In some embodiments, an isolated antibody that specifically binds gp41includes a heavy chain including amino acids 26-33 (27-38 in FIG. 6B)(CDR1), amino acids 51-60 (56-65 in FIG. 6B) (CDR2), and/or 99-120(105-126 in FIG. 6B) (CDR3) of SEQ ID NO: 1, and a light chain includingamino acids 26-31 (27-38 in FIG. 6B) (CDR1), 49-51 (56-65 in FIG. 6B)(CDR2), and/or 88-99 (105-116 in FIG. 6B) (CDR3) of SEQ ID NO: 2. Insome embodiments, an isolated antibody that specifically binds gp41includes a heavy chain including amino acids 26-33 (27-38 in FIG. 6B)(CDR1), amino acids 51-60 (56-65 in FIG. 6B) (CDR2), and/or 99-120(105-126 in FIG. 6B) (CDR3) of SEQ ID NO: 154, and a light chainincluding amino acids 26-31 (27-38 in FIG. 6B) (CDR1), 49-51 (56-65 inFIG. 6B) (CDR2), and/or 88-99 (105-116 in FIG. 6B) (CDR3) of SEQ ID NO:152. In some embodiments, an isolated antibody that specifically bindsgp41 includes a heavy chain including amino acids 26-33 (27-38 in FIG.6B) (CDR1), amino acids 51-60 (56-65 in FIG. 6B) (CDR2), and/or 99-120(105-126 in FIG. 6B) (CDR3) of SEQ ID NO: 192, and a light chainincluding amino acids 26-31 (27-38 in FIG. 6B) (CDR1), 49-51 (56-65 inFIG. 6B) (CDR2), and/or 88-99 (105-116 in FIG. 6B) (CDR3) of SEQ ID NO:152.

In additional examples, an isolated antibody that specifically bindsgp41 and is neutralizing includes a heavy chain variable region and alight chain variable region, wherein the heavy chain variable regionincludes the amino acid sequence set forth as one of SEQ ID NOs: 1, 3,5, 11, 146, 147-149, 187, 189-192, or 200-204, and the light chainvariable region includes the amino acid sequence set forth as one of SEQID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199. In one example,the heavy chain variable region includes the amino acid sequence setforth as SEQ ID NO: 1, and the light chain variable region includes theamino acid sequence set forth as SEQ ID NO: 2. In another example, theheavy chain variable region includes the amino acid sequence set forthas SEQ ID NO: 192, and the light chain variable region includes theamino acid sequence set forth as SEQ ID NO: 152. In a further example,the heavy chain variable region includes the amino acid sequence setforth as SEQ ID NO: 154, and the light chain variable region includesthe amino acid sequence set forth as SEQ ID NO: 152.

In further embodiments, an isolated antibody that specifically bindsgp41 includes a heavy chain variable region including an amino acidsequence including no more than 10 (such as more than 1, 2, 3, 4, 5, 6,7, 8, or no more than 9) amino acid substitutions compared to one of SEQID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204. Inadditional embodiments, an isolated antibody that specifically bindsgp41 includes a light chain variable region including an amino acidsequence including no more than 10 (such as more than 1, 2, 3, 4, 5, 6,7, 8, or no more than 9) amino acid substitutions compared to one of SEQID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199. In otherembodiments, an isolated antibody that specifically binds gp41 includesa heavy chain variable region including an amino acid sequence includingno more than 10 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, or no morethan 9) amino acid substitutions compared to one of SEQ ID NOs: 1, 3, 5,11, 146, 147-149, 187, 189-192, and 200-204, and a light chain variableregion including an amino acid sequence including no more than 10 (suchas more than 1, 2, 3, 4, 5, 6, 7, 8, or no more than 9) amino acidsubstitutions compared to one of SEQ ID NOs: 2, 4, 6, 12, 150-152,164-186, 188, or 197-199.

In other embodiments, an isolated antibody that specifically binds gp41includes a heavy chain variable region including an amino acid sequenceincluding no more than 10 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, orno more than 9) amino acid substitutions compared to one of SEQ ID NO:1, and a light chain variable region including an amino acid sequenceincluding no more than 10 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, orno more than 9) amino acid substitutions compared to SEQ ID NO: 2.

In further embodiments, an isolated antibody that specifically bindsgp41 includes a heavy chain variable region including the amino acidsequence set forth as SEQ ID NO: 187, wherein the amino acid sequenceincludes no more than 25 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or no more than24) amino acid substitutions compared to SEQ ID NO: 1. In additionalembodiments, an isolated antibody that specifically binds gp41 includesa light chain variable region including the amino acid sequence setforth as SEQ ID NO: 188, wherein the amino acid sequence includes nomore than 33 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 35, 36, 37, 38, 39, 30,31, 32 or no more than 33) amino acid substitutions compared to SEQ IDNO: 2. In other embodiments, an isolated antibody that specificallybinds gp41 includes a heavy chain including the amino acid sequence setforth as SEQ ID NO: 187, wherein the amino acid sequence includes nomore than 25 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or no more than 24) aminoacid substitutions compared to SEQ ID NO: 1, and a light chain includingthe amino acid sequence set forth as SEQ ID NO: 188, wherein the aminoacid sequence includes no more than 33 (such as more than 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,35, 36, 37, 38, 39, 30, 31, 32 or no more than 33) amino acidsubstitutions compared to SEQ ID NO: 2.

In further embodiments, an isolated antibody that specifically bindsgp41 includes a heavy chain variable region including an amino acidsequence having no more than 10 (such as more than 1, 2, 3, 4, 5, 6, 7,8, or no more than 9) amino acid substitutions compared to one of SEQ IDNOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204, and whereinthe substitutions are selected from the amino acid substitutions listedin FIGS. 60A and 60B. In additional embodiments, an isolated antibodythat specifically binds gp41 includes a light chain variable regionincluding an amino acid sequence including no more than 10 (such as morethan 1, 2, 3, 4, 5, 6, 7, 8, or no more than 9) amino acid substitutionscompared to one of the amino acid sequence set forth as one of SEQ IDNOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199, wherein the, andwherein the substitutions are selected from the amino acid substitutionsshown in FIGS. 61A and 61B. In other embodiments, an isolated antibodythat specifically binds gp41 includes a heavy chain variable regionincluding an amino acid sequence having no more than 10 (such as morethan 1, 2, 3, 4, 5, 6, 7, 8, or no more than 9) amino acid substitutionscompared to one of SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192,and 200-204, wherein the substitutions are selected from the amino acidsubstitutions shown in FIGS. 60A and 60B, and a light chain variableregion including an amino acid sequence including no more than 10 (suchas more than 1, 2, 3, 4, 5, 6, 7, 8, or no more than 9) amino acidsubstitutions compared to one of the amino acid sequence set forth asone of SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199, andwherein the substitutions are selected from the amino acid substitutionslisted in FIGS. 61A and 61B. In other embodiments, an isolated antibodythat specifically binds gp41 includes a heavy chain variable regionincluding an amino acid sequence having no more than 10 (such as morethan 1, 2, 3, 4, 5, 6, 7, 8, or no more than 9) amino acid substitutionscompared to SEQ ID NO: 1, wherein the substitutions are selected fromthe amino acid substitutions listed in FIGS. 60A and 60B, and a lightchain variable region including an amino acid sequence having no morethan 10 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, or no more than 9)amino acid substitutions compared to SEQ ID NO: 2, and wherein thesubstitutions are selected from the amino acid substitutions listed inFIGS. 61A and 61B.

In further embodiments, an isolated antibody that specifically bindsgp41 includes a heavy chain with at most one, at most two, at most threeor at most four amino acid substitutions in amino acids 26-31 (CDR1),49-51 (CDR2), and 87-98 (CDR3) of SEQ ID NO: 11, and a light chain. Insome embodiments, an isolated antibody that specifically binds gp41includes a heavy chain with at most one, at most two, at most three, atmost four amino acid or at most five amino acid substitutions in aminoacids 26-33 (CDR1), 51-60 (CDR2), and 99-120 (CDR3) of SEQ ID NO: 1.

In some embodiments, the antibody can include a heavy chain with at mostone, at most two, at most three, at most four amino acid or at most fiveamino acid substitutions in amino acids 26-33 (CDR1), 51-60 (CDR2), and99-120 (CDR3) of SEQ ID NO: 3. In some embodiments, the antibody caninclude a heavy chain with at most one, at most two, at most three, atmost four amino acid or at most five amino acid substitutions in aminoacids 26-33 (CDR1), 51-60 (CDR2), and 99-120 (CDR3) of SEQ ID NO: 5. Insome embodiments, the antibody can include a heavy chain with at mostone, at most two, at most three, at most four amino acid or at most fiveamino acid substitutions in amino acids 26-33 (CDR1), 51-60 (CDR2), and99-120 (CDR3) of SEQ ID NO: 154.

In further embodiments, an isolated antibody that specifically bindsgp41, and includes a light chain with at most one, at most two, at mostthree or at most four amino acid substitutions in amino acids 26-31(CDR1), 49-51 (CDR2), and 88-99 (CDR3) of SEQ ID NO: 12. In someembodiments, an isolated antibody that specifically binds gp41, andincludes a light chain with at most one, at most two, at most three orat most four amino acid substitutions in amino acids 26-31 (CDR1), 49-51(CDR2), and 88-99 (CDR3) of SEQ ID NO: 2. In some embodiments, theantibody can include a light chain with at most one, at most two, atmost three or at most four amino acid substitutions in amino acids 26-31(CDR1), 49-51 (CDR2), and 88-99 (CDR3) of SEQ ID NO: 4. In someembodiments, the antibody can include a light chain with at most one, atmost two, at most three or at most four amino acid substitutions inamino acids 26-31 (CDR1), 49-51 (CDR2), and 88-99 (CDR3) of SEQ ID NO:6. In some embodiments, the antibody can include a light chain with atmost one, at most two, at most three or at most four amino acidsubstitutions in amino acids 26-31 (CDR1), 49-51 (CDR2), and 88-99(CDR3) of SEQ ID NO: 152.

In some embodiments, an isolated antibody that specifically binds gp41as disclosed herein includes up to 10 amino acid substitutions (such asup to 1, 2, 3, 4, 5, 6, 7, 8, or up to 9 amino acid substitutions) inthe framework regions (for example, according to the Kabat, Clothia orIMGT numbering systems) of the heavy chain of the antibody, the lightchain of the antibody, or the heavy and light chains of the antibody.

In some embodiments, an isolated antibody that specifically binds gp41includes a heavy chain variable region including no more than 10 (suchas 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions in theframework regions of one of SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187,189-192, and 200-204. Framework regions of SEQ ID NOs: 1, 3, 5, 11, 146,147-149, 187, 189-192, and 200-204 include amino acids 1-25 (FR1), 34-50(FR2), 61-66 (FR3) and 121-131 (FR4) of SEQ ID NOs: 1, 3, 5, 11, 146,147-149, 187, 189-192, and 200-204, respectively (according to Kabatnumbering). In some embodiments, an isolated antibody that specificallybinds gp41 includes a light chain variable region including no more than10 (such as 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions in theframework regions of SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or197-199, respectively. Framework regions of SEQ ID NO: 2 include aminoacids 1-25 (LFR2), 32-48 (LFR2), 52-87 (LFR3) and 99-108 (OFR4) of SEQID NO: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199, respectively(according to Kabat numbering).

In further embodiments, an isolated antibody that specifically bindsgp41 includes a heavy chain variable region including no more than 10(such as 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions in theframework regions of SEQ ID NO: 1 and a light chain variable regionincluding no more than 10 (such as 1, 2, 3, 4, 5, 6, 7, 8 or 9) aminoacid substitutions in the framework regions of SEQ ID NOs: 2. In furtherembodiments, an isolated antibody that specifically binds gp41 includesa heavy chain variable region including no more than 10 (such as 1, 2,3, 4, 5, 6, 7, 8 or 9) amino acid substitutions in the framework regionsof SEQ ID NO: 154 and a light chain variable region including no morethan 10 (such as 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutionsin the framework regions of SEQ ID NO: 152. In further embodiments, anisolated antibody that specifically binds gp41 includes a heavy chainvariable region including no more than 10 (such as 1, 2, 3, 4, 5, 6, 7,8 or 9) amino acid substitutions in the framework regions of SEQ ID NO:192 and a light chain variable region including no more than 10 (such as1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions in the frameworkregions of SEQ ID NO: 152.

In some embodiments, the heavy chain of the human monoclonal antibodyincludes an amino acid sequence having at least 80% (such as at least85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%)sequence identity with the amino acid sequence set forth as one of SEQID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, or 200-204. Inadditional examples, the heavy chain includes the amino acid sequenceset forth as one of SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192,or 200-204. In some examples, the light chain of the human monoclonalantibody includes the amino acid sequence having at least 80% (such asat least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least99%) sequence identity with the amino acid sequence set forth as one ofSEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199. In furtherembodiments, an isolated antibody that specifically binds gp41 includesa heavy chain variable region and a light chain variable region, whereinthe heavy chain variable region includes the amino acid sequence havingat least 80% (such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or at least 99%) sequence identity with the amino acidsequence set forth as one of SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187,189-192, or 200-204, and the light chain variable region includes theamino acid sequence having at least 80% (such as at least 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%) sequence identitywith the amino acid sequence set forth as one of SEQ ID NOs: 2, 4, 6,12, 150-152, 164-186, 188, or 197-199.

As disclosed herein, deep sequencing was used to identify additionalantibodies that bind to substantially similar epitopes on the surface ofgp41 in substantially the same orientation that 10E8, 7H6, and/or 7N16bind. Exemplary nucleic acid sequences encoding antibody heavy chainsare set forth as SEQ ID NOs: 35-115 in the accompanying sequencelisting. These encode antibody heavy chain variable regions at leastabout 80% identical to the 10E8 antibody heavy chain variable region(SEQ ID NO: 1). Thus, disclosed herein are nucleic acid moleculesencoding antibody heavy chain variable regions that are at least about80% identical to the heavy chain variable region set forth as SEQ Id NO:1, such as at least about 80%, at least about 81%, at least about 82%,at least about 83%, at least about 84%, at least about 85% at leastabout 86%, at least about 87%, at least about 88%, or even at leastabout 89% at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95% at least about96%, at least about 97%, at least about 98%, or even at least about 99%identical to SEQ ID NO: 1. Exemplary nucleic acid sequences encoding10E8-like antibody light chains are set forth as SEQ ID NOs: 116-145 inthe accompanying sequence listing. Exemplary nucleic acid sequencesencoding antibody light chains are set forth as SEQ ID NOs: 116-145 inthe accompanying sequence listing. These encode antibody light chainvariable regions at least about 80% identical to the 10E8 antibody lightchain variable region (SEQ ID NO: 2). Thus, disclosed herein are nucleicacid molecules encoding antibody light chain variable regions that areat least about 80% identical to the heavy chain variable region setforth as SEQ ID NO: 2, such as at least about 80%, at least about 81%,at least about 82%, at least about 83%, at least about 84%, at leastabout 85% at least about 86%, at least about 87%, at least about 88%, oreven at least about 89% at least about 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95% atleast about 96%, at least about 97%, at least about 98%, or even atleast about 99% identical to SEQ ID NO: 2.

In some embodiments, an isolated antibody that specifically binds gp41includes one or more of the heavy chain complementarity determiningregions (CDRs) encoded by the nucleic acid sequence set forth as one ofSEQ ID NOs: 35-115. In some embodiments, an isolated human monoclonalantibody that specifically binds gp41 includes a heavy chain withal ofthe CDRs encoded by the nucleic acid sequence set forth as one of SEQ IDNOs: 35-115. In specific examples, the heavy chain of the humanmonoclonal antibody includes the amino acid sequence encoded by thenucleic acid sequence set forth as one of SEQ ID NOs: 35-115.

In some embodiments, an isolated antibody that specifically binds gp41includes one or more of the light chain complementarity determiningregions (CDRs) encoded by the nucleic acid sequence set forth as one ofSEQ ID NOs: 116-145. In some embodiments, an isolated human monoclonalantibody that specifically binds gp41 includes a light chain with all ofthe CDRs antibody encoded by the nucleic acid sequence set forth as oneof SEQ ID NOs: 116-145. In specific examples, the light chain of thehuman monoclonal antibody includes the amino acid sequence encoded bythe nucleic acid sequence set forth as one of SEQ ID NOs: 116-145.

In some embodiments, an isolated antibody that specifically binds gp41includes a heavy chain variable region encoded by a nucleic acid derivedfrom the IGHV3-15 germline allelic origin, for example the IGHV3-15*01,IGHV3-15*02, IGHV3-15*03, IGHV3-15*04, IGHV3-15*05, IGHV3-15*06,IGHV3-15*07, IGHV3-15*08, IGHV3-15*09, IGHV3-15*10, IGHV3-15*11,IGHV3-15*12, IGHV3-15*13, IGHV3-15*14, or IGHV3-15*15 germline allelicorigin. In some embodiments, the heavy chain variable region is encodedby a nucleic acid derived from the IGHV3-15 germline allelic origin, forexample the IGHV3-15*01, IGHV3-15*02, IGHV3-15*03, IGHV3-15*04,IGHV3-15*05, IGHV3-15*06, IGHV3-15*07, IGHV3-15*08, IGHV3-15*09,IGHV3-15*10, IGHV3-15*11, IGHV3-15*12, IGHV3-15*13, IGHV3-15*14, orIGHV3-15*15 germline allelic origin, and is about 10%, 15%, 20%, 25%,30%, 35% or 40%, such as about 15% to 40% divergent from the respectiveheavy chain germline sequence.

In some embodiments, an isolated antibody that specifically binds gp41includes a light chain variable region encoded by a nucleic acid derivedfrom the IGLV3-19 germline allelic origin, such as an IGLV3-19*01germline allelic origin. In some embodiments, the light chain is encodedby a nucleic acid derived from the IGLV3-19 germline allelic origin,such as an IGLV3-19*01 germline allelic origin, and is about 10%, 15%,20%, 25%, 30%, 35% or 40%, such as about 15% to 40% divergent from therespective light chain germline sequence.

In some examples, the heavy chain variable domain is a clonal variantfrom donor N152, with a heavy chain encoded by VH3-15 gene and VJ-1 Jgenes. In other examples, the light chain variable domain is a clonalvariant from donor N152, with a light chain encoded by a LV3-19 V geneand a LJ-3 J gene. The isolated monoclonal antibody can include a heavychain and a light chain, wherein the heavy chain variable region is aclonal variant from donor N152 with a heavy chain variable region aminoacid sequence set forth as SEQ ID NO: 1. The a heavy chain is derivedfrom a VH3-15 gene and LJ-3 J genes. The light chain variable domain isa clonal variant from donor N152 with a light chain variable regionamino acid sequence set forth as SEQ ID NO: 2. The light chain isderived a LV3-19 V gene and a LJ-3 J gene, the monoclonal antibodyspecifically binds gp41, competes with 10E8 for binding to gp41, and isneutralizing

In some embodiments, the heavy chain of a 10E8-like antibody can becomplemented by the light chain of the 10E8, 7H6, and/or 7N16 antibodyand still retain binding for gp41, for example retain specific bindingfor the 10E8 epitope. In some embodiments, the light chain of a10E8-like antibody can be complemented by the heavy chain of the 10E8,7H6, and/or 7N16 antibody and still retain binding for gp41, for exampleretain specific binding for the 10E8 epitope. Thus, disclosed herein are10E8-like antibodies that can be identified by complementation of theheavy or light chains of 10E8, 7H6, and/or 7N16.

Once a heavy or light chain variable domain of interest is identified,binding to gp41 or an epitope of interest (such as the 10E8 epitope) canbe determined using a cross complementation analysis. Briefly, if thevariable domain of interest is a heavy chain variable domain, the aminoacid sequence of this heavy chain variable domain is produced. The heavychain variable domain is then paired with a reference sequence lightchain variable domain, such as 10E8 (SEQ ID NO: 2), 7H6 (SEQ ID NO: 4),and/or 7N16 (SEQ ID NO: 6), light chain variable domain, and it isdetermined if the antibody specifically binds the antigen (or epitope)with a specified affinity, such as a K_(D) of 10⁻⁸, 10⁻⁹ or 10⁻¹⁰.Similarly, if the variable domain of interest is a light chain variabledomain, this amino acid sequence is produced. The variable light chainvariable domain is then paired with a reference sequence heavy chainvariable domain, such as variable domain is then paired with a referencesequence light chain variable domain, such as 10E8 (SEQ ID NO: 1), 7H6(SEQ ID NO: 3), and/or 7N16 (SEQ Id NO: 5) heavy chain variable domain,and it is determined if the antibody specifically binds the antigen (orepitope) with a specified affinity, such as a K_(D) of 10⁻⁸, 10⁻⁹ or10⁻¹⁰.

Fully human monoclonal antibodies include human framework regions. Thus,any of the antibodies that specifically bind gp41 herein can include thehuman framework region and can include the framework regions of theamino acid sequence set forth as one of SEQ ID NO; 1-6, 11, 12, and/or146-192 or encoded by one of SEQ ID NOs: 35-145. However, the frameworkregions can be from another source. Additional examples of frameworksequences that can be used include the amino acid framework sequences ofthe heavy and light chains disclosed in PCT Publication No. WO2006/074071 (see, for example, SEQ ID NOs: 1-16), which is hereinincorporated by reference.

In some embodiments, one or more of the heavy and/or light chaincomplementarity determining regions (CDRs) from gp41 antibody set forthone of 1-6, 11, 12, and/or 146-192 or encoded by SEQ ID NOs: 35-145 isexpressed on the surface of another protein, such as a scaffold protein.The expression of domains of antibodies on the surface of a scaffoldingprotein are known in the art (see e.g. Liu et al., J. Virology 85(17):8467-8476, 2011). Such expression creates a chimeric protein thatretains the binding for gp41, such as specific binding to the 10E8epitope. As described in Example 1, the 10E8 class of antibodies makesmost of its contacts through the heavy chain CDRs (see, for example, thetables given as FIGS. 27-30, and the molecular models shown in FIGS. 4,5, 12, 15, 16 and 39-41A). Thus, in some specific embodiments, one ormore of the heavy chain CDRs is grafted onto a scaffold protein, such asone or more of heavy chain CDR1, CDR2, and/or CDR3 set forth as one ofSEQ ID NO; 1, 3, 5, 11, 146-149, 153-163, 187, 189-192, or 200-204 orencoded by one of SEQ ID NOs: 35-115.

The monoclonal antibody can be of any isotype. The monoclonal antibodycan be, for example, an IgM or an IgG antibody, such as IgG₁, IgG₂,IgG₃, or IgG₄. The class of an antibody that specifically binds gp41 canbe switched with another. In one aspect, a nucleic acid moleculeencoding V_(L) or V_(H) is isolated using methods well-known in the art,such that it does not include any nucleic acid sequences encoding theconstant region of the light or heavy chain, respectively.

In particular examples, the V_(H) amino acid sequence is set forth asone of SEQ ID NO: 1, 3, 5, 11, 146-149, 153-163, 187, 189-192, or200-204 or encoded by one of SEQ ID NOs: 35-115. In other examples, theV_(L) amino acid sequence is set forth as one of SEQ ID NO: 2, 4, 6, 12,150-152, 164-186, 188, or 197-199 or encoded by one of SEQ ID NOs:116-145. The nucleic acid molecule encoding V_(L) or V_(H) is thenoperatively linked to a nucleic acid sequence encoding a C_(L) or C_(H)from a different class of immunoglobulin molecule. This can be achievedusing a vector or nucleic acid molecule that includes a C_(L) or C_(H)chain, as known in the art. For example, an antibody that specificallybinds gp41, that was originally IgM may be class switched to an IgG.Class switching can be used to convert one IgG subclass to another, suchas from IgG₁ to IgG₂, IgG₃, or IgG₄.

In some examples, the disclosed antibodies are oligomers of antibodies,such as dimers, trimers, tetramers, pentamers, hexamers, septamers,octomers and so on. In some examples, the antibodies are pentamers.

In some examples, the antibodies, or an antibody binding fragmentthereof is modified such that it is directly cytotoxic to infectedcells, or uses natural defenses such as complement, antibody dependentcellular cytotoxicity (ADCC), or phagocytosis by macrophages.

Antibody fragments are encompassed by the present disclosure, such asFab, F(ab′)₂, and Fv which include a heavy chain and light chainvariable region and specifically bind gp41. These antibody fragmentsretain the ability to selectively bind with the antigen and are“antigen-binding” fragments. These fragments include:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains; and

(5) Single chain antibody (such as scFv), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

(6) A dimer of a single chain antibody (scFV₂), defined as a dimer of ascFV. This has also been termed a “miniantibody.”

Methods of making these fragments are known in the art (see for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988).

In a further group of embodiments, the antibodies are Fv antibodies,which are typically about 25 kDa and contain a complete antigen-bindingsite with three CDRs per each heavy chain and each light chain. Toproduce these antibodies, the V_(H) and the V_(L) can be expressed fromtwo individual nucleic acid constructs in a host cell. In particularexamples, the V_(H) amino acid sequence includes the CDRs from one ofSEQ ID NOs: 1, 3, 5, 11, 146-149, 153-163, 187 or 189-192 or encoded byone of SEQ ID NOs: 35-115. In other examples, the V_(L) amino acidsequence includes the CDRs from SEQ ID NOs: 2, 4, 6, 12, 150-152,164-186, 188, or 197-199 or encoded by one of SEQ ID NOs: 116-145. Inadditional examples, the V_(H) amino acid sequence includes the aminoacid sequence set forth as one of SEQ ID NOs: 1, 3, 5, 11, 146-149,153-163, 187 or 189-192 or encoded by one of SEQ ID NOs: 35-115. Inother examples, the V_(L) amino acid sequence includes the amino acidsequence set forth as SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or197-199 or encoded by one of SEQ ID NOs: 116-145.

If the V_(H) and the V_(L) are expressed non-contiguously, the chains ofthe Fv antibody are typically held together by noncovalent interactions.However, these chains tend to dissociate upon dilution, so methods havebeen developed to crosslink the chains through glutaraldehyde,intermolecular disulfides, or a peptide linker. Thus, in one example,the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chainvariable region and the light chain variable region are chemicallylinked by disulfide bonds.

In an additional example, the Fv fragments include V_(H) and V_(L)chains connected by a peptide linker. These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural gene includingDNA sequences encoding the V_(H) and V_(L) domains connected by anoligonucleotide. The structural gene is inserted into an expressionvector, which is subsequently introduced into a host cell such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are known in the art (see Whitlow et al., Methods: a Companion toMethods in Enzymology, Vol. 2, page 97, 1991; Bird et al., Science242:423, 1988; U.S. Pat. No. 4,946,778; Pack et al., Bio/Technology11:1271, 1993; and Sandhu, supra). Dimers of a single chain antibody(scFV₂), are also contemplated.

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli of DNA encoding the fragment.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly (see U.S. Pat. Nos. 4,036,945 and 4,331,647,and references contained therein; Nisonhoff et al., Arch. Biochem.Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al.,Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; andColigan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

One of skill will realize that conservative variants of the antibodiescan be produced. Such conservative variants employed in antibodyfragments, such as dsFv fragments or in scFv fragments, will retaincritical amino acid residues necessary for correct folding andstabilizing between the V_(H) and the V_(L) regions, and will retain thecharge characteristics of the residues in order to preserve the low pIand low toxicity of the molecules. Amino acid substitutions (such as atmost one, at most two, at most three, at most four, or at most fiveamino acid substitutions) can be made in the V_(H) and the V_(L) regionsto increase yield. In particular examples, the V_(H) sequence is SEQ IDNOs: 1, 3, 5, 11, 146-149, 153-163, 187 or 189-192 or encoded by one ofSEQ ID NOs: 35-115. In other examples, the V_(L) sequence is SEQ ID NOs:2, 4, 6, 12, 150-152, 164-186, 188, or 197-199 or encoded by one of SEQID NOs: 116-145. Conservative amino acid substitution tables providingfunctionally similar amino acids are well known to one of ordinary skillin the art. The following six groups are examples of amino acids thatare considered to be conservative substitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The antibodies disclosed herein can be isolated using cloaked antigens,as described in PCT Publication No. WO 2009/100376. Briefly, antigensare cloaked to target antigenicity of the antigen to a specific epitopethat specifically bound by the antibody of interest, such as aneutralizing antibody.

Additional recombinant human neutralizing antibodies that specificallybind the same epitope of gp41 bound by the antibodies disclosed hereinthat specifically bind gp41, can be isolated by screening of arecombinant combinatorial antibody library, such as a Fab phage displaylibrary (see, for example, U.S. Patent Application Publication No.2005/0123900). In some cases the phage display libraries are preparedusing cDNAs of the variable regions of heavy and light chains preparedfrom mRNA derived from human lymphocytes. Methodologies for preparingand screening such libraries are known in the art. There arecommercially available kits for generating phage display libraries (forexample, the Pharmacia Recombinant Phage Antibody System, Catalog No.27-9400-01; and the Stratagene SurfZAP™ phage display kit, catalog no.240612). There are also other methods and reagents that can be used ingenerating and screening antibody display libraries (see, for example,U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCTPublication No. WO 91/17271; PCT Publication No. WO 92/20791; PCTPublication No. WO 92/15679; PCT Publication No. WO 93/01288; PCTPublication No. WO 92/01047; PCT Publication No. WO 92/09690; Fuchs etal., Bio/Technology 9:1370-1372, 1991; Hay et al., Hum. Antibod.Hybridomas 3:81-85, 1992; Huse et al., Science 246:1275-1281, 1989;McCafferty et al., Nature 348:552-554, 1990; Griffiths et al., EMBO J.12:725-734, 1993)

In one embodiment, to isolate additional human antibodies thatspecifically bind gp41, a neutralizing antibody that specifically bindsgp41, as described herein, is first used to select human heavy and lightchain sequences having similar binding activity toward gp41, such asusing the epitope imprinting methods disclosed in PCT Publication No. WO93/06213. The antibody libraries used in this method are scFv librariesprepared and screened, using methods such as those as described in PCTPublication No. WO 92/01047, McCafferty et al., Nature 348:552-554,1990; and/or Griffiths et al., EMBO J. 12:725-734, 1993 using gp120.

Once initial human variable light chain (V_(L)) and variable heavy chain(V_(H)) segments are selected, “mix and match” experiments, in whichdifferent pairs of the initially selected V_(L) and V_(H) segments arescreened for gp41 binding are performed to select V_(L)/V_(H) paircombinations of interest. Additionally, to increase binding affinity ofthe antibody, the V_(L) and V_(H) segments can be randomly mutated, suchas within H-CDR3 region or the L-CDR3 region, in a process analogous tothe in vivo somatic mutation process responsible for affinity maturationof antibodies during a natural immune response. Thus in vitro affinitymaturation can be accomplished by amplifying V_(H) and V_(L) regionsusing PCR primers complementary to the H-CDR3 or L-CDR3, respectively.In this process, the primers have been “spiked” with a random mixture ofthe four nucleotide bases at certain positions such that the resultantPCR products encode V_(H) and V_(L) segments into which random mutationshave been introduced into the V_(H) and/or V_(L) CDR3 regions. Theserandomly mutated V_(H) and V_(L) segments can be tested to determine thebinding affinity for gp41. In particular examples, the V_(H) amino acidsequence is SEQ ID NOs: 1, 3, 5, or 11, 146-149, 153-163, 187, 189-192,or 200-204 or encoded by one of SEQ ID NOs: 35-115. In other examples,the V_(L) amino acid sequence is SEQ ID NOs: 2, 4, 6, 12, 150-152,164-186, 188, or 197-199 or encoded by one of SEQ ID NOs: 116-145.

Following screening and isolation of an antibody that binds gp41 from arecombinant immunoglobulin display library, nucleic acid encoding theselected antibody can be recovered from the display package (forexample, from the phage genome) and subcloned into other expressionvectors by standard recombinant DNA techniques, as described herein. Ifdesired, the nucleic acid can be further manipulated to create otherantibody fragments, also as described herein. To express a recombinantantibody isolated by screening of a combinatorial library, the DNAencoding the antibody is cloned into a recombinant expression vector andintroduced into a mammalian host cells, as described herein.

Effector molecules, such as therapeutic, diagnostic, or detectionmoieties can be linked to an antibody of interest, using any number ofmeans known to those of skill in the art. Both covalent and noncovalentattachment means may be used. The procedure for attaching an effectormolecule to an antibody varies according to the chemical structure ofthe effector. Polypeptides typically contain a variety of functionalgroups; such as carboxylic acid (COOH), free amine (—NH₂) or sulfhydryl(—SH) groups, which are available for reaction with a suitablefunctional group on an antibody to result in the binding of the effectormolecule. Alternatively, the antibody is derivatized to expose or attachadditional reactive functional groups. The derivatization may involveattachment of any of a number of linker molecules such as thoseavailable from Pierce Chemical Company, Rockford, Ill. The linker can beany molecule used to join the antibody to the effector molecule. Thelinker is capable of forming covalent bonds to both the antibody and tothe effector molecule. Suitable linkers are well known to those of skillin the art and include, but are not limited to, straight orbranched-chain carbon linkers, heterocyclic carbon linkers, or peptidelinkers. Where the antibody and the effector molecule are polypeptides,the linkers may be joined to the constituent amino acids through theirside groups (such as through a disulfide linkage to cysteine) or to thealpha carbon amino and carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will includelinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site.

In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, label (such as enzymes or fluorescent molecules) drugs,toxins, and other agents to antibodies one skilled in the art will beable to determine a suitable method for attaching a given agent to anantibody or other polypeptide.

The antibodies or antibody fragments disclosed herein can be derivatizedor linked to another molecule (such as another peptide or protein). Ingeneral, the antibody or portion thereof is derivatized such that thebinding to gp41 is not affected adversely by the derivatization orlabeling. For example, the antibody can be functionally linked (bychemical coupling, genetic fusion, noncovalent association or otherwise)to one or more other molecular entities, such as another antibody (forexample, a bispecific antibody or a diabody), a detection agent, apharmaceutical agent, and/or a protein or peptide that can mediateassociate of the antibody or antibody portion with another molecule(such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by cross-linking two ormore antibodies (of the same type or of different types, such as tocreate bispecific antibodies). Suitable crosslinkers include those thatare heterobifunctional, having two distinctly reactive groups separatedby an appropriate spacer (such asm-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (suchas disuccinimidyl suberate). Such linkers are available from PierceChemical Company (Rockford, Ill.).

An antibody that specifically binds gp41 can be labeled with adetectable moiety. Useful detection agents include fluorescentcompounds, including fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. Bioluminescent markers are also of use, such asluciferase, Green fluorescent protein, Yellow fluorescent protein. Anantibody can also be labeled with enzymes that are useful for detection,such as horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase, glucose oxidase and the like. When an antibody is labeledwith a detectable enzyme, it can be detected by adding additionalreagents that the enzyme uses to produce a reaction product that can bediscerned. For example, when the agent horseradish peroxidase is presentthe addition of hydrogen peroxide and diaminobenzidine leads to acolored reaction product, which is visually detectable. An antibody mayalso be labeled with biotin, and detected through indirect measurementof avidin or streptavidin binding. It should be noted that the avidinitself can be labeled with an enzyme or a fluorescent label.

An antibody may be labeled with a magnetic agent, such as gadolinium.Antibodies can also be labeled with lanthanides (such as europium anddysprosium), and manganese. Paramagnetic particles such assuperparamagnetic iron oxide are also of use as labels. An antibody mayalso be labeled with a predetermined polypeptide epitopes recognized bya secondary reporter (such as leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags). Insome embodiments, labels are attached by spacer arms of various lengthsto reduce potential steric hindrance.

An antibody can also be labeled with a radiolabeled amino acid. Theradiolabel may be used for both diagnostic and therapeutic purposes.Examples of labels for polypeptides include, but are not limited to, thefollowing radioisotopes or radionucleotides: ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I.

An antibody can also be derivatized with a chemical group such aspolyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrategroup. These groups may be useful to improve the biologicalcharacteristics of the antibody, such as to increase serum half-life orto increase tissue binding.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

The present disclosure also relates to the crystals obtained from the10E8, 7H6, and/or 7N16, antibody or portions thereof in complex withgp41 (or gp41 peptides), the crystal structures of the 10E8, 7H6, and/or7N16 antibody or portions thereof in complex with gp41 (or gp41peptides), the three-dimensional coordinates of the 10E8, 7H6, and/or7N16 antibody or portions thereof in complex with gp41 (or gp41peptides) and three-dimensional structures of models of the 10E8, 7H6,and/or 7N16 antibody or portions thereof in complex with gp41 (or gp41peptides).

Those of skill in the art will understand that a set of structurecoordinates for the 10E8, 7H6, and/or 7N16 antibody or portions thereofin complex with gp41 or a portion thereof, is a relative set of pointsthat define a shape in three dimensions. Thus, it is possible that anentirely different set of coordinates could define a similar oridentical shape. Moreover, slight variations in the individualcoordinates will have little effect on overall shape. The variations incoordinates discussed above may be generated because of mathematicalmanipulations of the structure coordinates.

This disclosure further provides systems, such as computer systems,intended to generate structures and/or perform rational drug or compounddesign for an antigenic compound capable of eliciting an immune responsein a subject. The system can contain one or more or all of: atomicco-ordinate data according to 10E8, 7H6, and/or 7N16 antibody complex ora subset thereof, and the figures derived therefrom by homologymodeling, the data defining the three-dimensional structure of a 10E8,7H6, and/or 7N16 antibody complex or at least one sub-domain thereof, orstructure factor data for gp41, the structure factor data beingderivable from the atomic co-ordinate data of 10E8, 7H6, and/or 7N16antibody complex or a subset thereof and the figures.

B. Polynucleotides and Expression

Nucleic acid molecules (also referred to as polynucleotides) encodingthe polypeptides provided herein (including, but not limited toantibodies) can readily be produced by one of skill in the art. Forexample, these nucleic acids can be produced using the amino acidsequences provided herein (such as the CDR sequences, heavy chain andlight chain sequences).

One of skill in the art can readily use the genetic code to construct avariety of functionally equivalent nucleic acids, such as nucleic acidswhich differ in sequence but which encode the same antibody sequence, orencode a conjugate or fusion protein including the V_(L) and/or V_(H)nucleic acid sequence.

Nucleic acid sequences encoding the antibodies that specifically bindgp41 can be prepared by any suitable method including, for example,cloning of appropriate sequences or by direct chemical synthesis bymethods such as the phosphotriester method of Narang et al., Meth.Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al.,Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramidite method ofBeaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solid phasephosphoramidite triester method described by Beaucage & Caruthers,Tetra. Letts. 22(20):1859-1862, 1981, for example, using an automatedsynthesizer as described in, for example, Needham-VanDevanter et al.,Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method ofU.S. Pat. No. 4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is generally limited tosequences of about 100 bases, longer sequences may be obtained by theligation of shorter sequences.

Exemplary nucleic acids can be prepared by cloning techniques. Examplesof appropriate cloning and sequencing techniques, and instructionssufficient to direct persons of skill through many cloning exercises arefound in Sambrook et al., supra, Berger and Kimmel (eds.), supra, andAusubel, supra. Product information from manufacturers of biologicalreagents and experimental equipment also provide useful information.Such manufacturers include the SIGMA Chemical Company (Saint Louis,Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway,N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem GenesCorp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc.,GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), FlukaChemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),Invitrogen (Carlsbad, Calif.), and Applied Biosystems (Foster City,Calif.), as well as many other commercial sources known to one of skill.

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

Any of the nucleic acids encoding any of the antibodies, V_(H) and/orV_(L), disclosed herein (or fragment thereof) can be expressed in arecombinantly engineered cell such as bacteria, plant, yeast, insect andmammalian cells. These antibodies can be expressed as individual V_(H)and/or V_(L) chain, or can be expressed as a fusion protein. Animmunoadhesin can also be expressed. Thus, in some examples, nucleicacids encoding a V_(H) and V_(L), and immunoadhesin are provided. Thenucleic acid sequences can optionally encode a leader sequence.

To create a single chain antibody, (scFv) the V_(H)- and V_(L)-encodingDNA fragments are operatively linked to another fragment encoding aflexible linker, e.g., encoding the amino acid sequence (G1Y₄-Ser)₃,such that the V_(H) and V_(L) sequences can be expressed as a contiguoussingle-chain protein, with the V_(L) and V_(H) domains joined by theflexible linker (see, e.g., Bird et al., Science 242:423-426, 1988;Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; McCaffertyet al., Nature 348:552-554, 1990). Optionally, a cleavage site can beincluded in a linker, such as a furin cleavage site.

The nucleic acid encoding the V_(H) and/or the V_(L) optionally canencode an Fc domain (immunoadhesin). The Fc domain can be an IgA, IgM orIgG Fc domain. The Fc domain can be an optimized Fc domain, as describedin U.S. Published Patent Application No. 20100/093979, incorporatedherein by reference. In one example, the immunoadhesin is an IgG₁ Fc. Inone example, the immunoadhesin is an IgG₃ Fc.

The single chain antibody may be monovalent, if only a single V_(H) andV_(L) are used, bivalent, if two V_(H) and V_(L) are used, orpolyvalent, if more than two V_(H) and V_(L) are used. Bispecific orpolyvalent antibodies may be generated that bind specifically to gp120and to another molecule, such as gp41. The encoded V_(H) and V_(L)optionally can include a furin cleavage site between the V_(H) and V_(L)domains.

It is expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of proteinsincluding E. coli, other bacterial hosts, yeast, and various highereukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.

The host cell can be a gram positive bacteria including, but are notlimited to, Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Methods for expressing protein in gram positivebacteria, such as Lactobaccillus are well known in the art, see forexample, U.S. Published Patent Application No. 20100/080774. Expressionvectors for lactobacillus are described, for example in U.S. Pat. Nos.6,100,388, and 5,728,571. Leader sequences can be included forexpression in Lactobacillus. Gram negative bacteria include, but notlimited to, E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, andUreaplasma.

One or more DNA sequences encoding the antibody or fragment thereof canbe expressed in vitro by

DNA transfer into a suitable host cell. The cell may be prokaryotic oreukaryotic. The term also includes any progeny of the subject host cell.It is understood that all progeny may not be identical to the parentalcell since there may be mutations that occur during replication. Methodsof stable transfer, meaning that the foreign DNA is continuouslymaintained in the host, are known in the art. Hybridomas expressing theantibodies of interest are also encompassed by this disclosure.

The expression of nucleic acids encoding the isolated proteins describedherein can be achieved by operably linking the DNA or cDNA to a promoter(which is either constitutive or inducible), followed by incorporationinto an expression cassette. The promoter can be any promoter ofinterest, including a cytomegalovirus promoter and a human T celllymphotrophic virus promoter (HTLV)-1. Optionally, an enhancer, such asa cytomegalovirus enhancer, is included in the construct. The cassettescan be suitable for replication and integration in either prokaryotes oreukaryotes. Typical expression cassettes contain specific sequencesuseful for regulation of the expression of the DNA encoding the protein.For example, the expression cassettes can include appropriate promoters,enhancers, transcription and translation terminators, initiationsequences, a start codon (i.e., ATG) in front of a protein-encodinggene, splicing signal for introns, sequences for the maintenance of thecorrect reading frame of that gene to permit proper translation of mRNA,and stop codons. The vector can encode a selectable marker, such as amarker encoding drug resistance (for example, ampicillin or tetracyclineresistance).

To obtain high level expression of a cloned gene, it is desirable toconstruct expression cassettes which contain, at the minimum, a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation (internal ribosomal binding sequences), and atranscription/translation terminator. For E. coli, this includes apromoter such as the T7, trp, lac, or lambda promoters, a ribosomebinding site, and preferably a transcription termination signal. Foreukaryotic cells, the control sequences can include a promoter and/or anenhancer derived from, for example, an immunoglobulin gene, HTLV, SV40or cytomegalovirus, and a polyadenylation sequence, and can furtherinclude splice donor and/or acceptor sequences (for example, CMV and/orHTLV splice acceptor and donor sequences). The cassettes can betransferred into the chosen host cell by well-known methods such astransformation or electroporation for E. coli and calcium phosphatetreatment, electroporation or lipofection for mammalian cells. Cellstransformed by the cassettes can be selected by resistance toantibiotics conferred by genes contained in the cassettes, such as theamp, gpt, neo and hyg genes.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the antibody,labeled antibody, or antibody binding fragment thereof, and a secondforeign DNA molecule encoding a selectable phenotype, such as the herpessimplex thymidine kinase gene. Another method is to use a eukaryoticviral vector, such as simian virus 40 (SV40) or bovine papilloma virus,to transiently infect or transform eukaryotic cells and express theprotein (see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982). One of skill in the art can readily usean expression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the COS,CHO, HeLa and myeloma cell lines.

Modifications can be made to a nucleic acid encoding a polypeptidedescribed herein without diminishing its biological activity. Somemodifications can be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, termination codons, a methionine added at the aminoterminus to provide an initiation, site, additional amino acids placedon either terminus to create conveniently located restriction sites, oradditional amino acids (such as poly His) to aid in purification steps.In addition to recombinant methods, the immunoconjugates, effectormoieties, and antibodies of the present disclosure can also beconstructed in whole or in part using standard peptide synthesis wellknown in the art.

Once expressed, the recombinant immunoconjugates, antibodies, and/oreffector molecules can be purified according to standard procedures ofthe art, including ammonium sulfate precipitation, affinity columns,column chromatography, and the like (see, generally, R. Scopes, PROTEINPURIFICATION, Springer-Verlag, N.Y., 1982). The antibodies,immunoconjugates and effector molecules need not be 100% pure. Oncepurified, partially or to homogeneity as desired, if to be usedtherapeutically, the polypeptides should be substantially free ofendotoxin.

Methods for expression of antibodies and/or refolding to an appropriateactive form, including single chain antibodies, from bacteria such as E.coli have been described and are well-known and are applicable to theantibodies disclosed herein. See, Buchner et al., Anal. Biochem.205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991; Huse et al.,Science 246:1275, 1989 and Ward et al., Nature 341:544, 1989.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena et al., Biochemistry 9: 5015-5021, 1970, andespecially as described by Buchner et al., supra.

Renaturation is typically accomplished by dilution (for example,100-fold) of the denatured and reduced protein into refolding buffer. Anexemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. An exemplary yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5-foldmolar excess of one protein over the other is not exceeded. Excessoxidized glutathione or other oxidizing low molecular weight compoundscan be added to the refolding solution after the redox-shuffling iscompleted.

In addition to recombinant methods, the antibodies, labeled antibodiesand antibody binding fragments thereof that are disclosed herein canalso be constructed in whole or in part using standard peptidesynthesis. Solid phase synthesis of the polypeptides of less than about50 amino acids in length can be accomplished by attaching the C-terminalamino acid of the sequence to an insoluble support followed bysequential addition of the remaining amino acids in the sequence.Techniques for solid phase synthesis are described by Barany &Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: SpecialMethods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J.Am. Chem. Soc. 85:2149-2156, 1963, and Stewart et al., Solid PhasePeptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984.Proteins of greater length may be synthesized by condensation of theamino and carboxyl termini of shorter fragments. Methods of formingpeptide bonds by activation of a carboxyl terminal end (such as by theuse of the coupling reagent N,N′-dicylohexylcarbodimide) are well knownin the art.

C. Compositions and Therapeutic Methods

Methods are disclosed herein for the prevention or treatment of an HIVinfection, such as an HIV-1 infection. Prevention can include inhibitionof infection with HIV-1. The methods include contacting a cell with aneffective amount of the human monoclonal antibodies disclosed hereinthat specifically binds gp41, or an antibody binding fragment thereof oran nucleic acid encoding such antibodies or antibody binding fragmentsthereof. The method can also include administering to a subject atherapeutically effective amount of the human monoclonal antibodies to asubject, or an antibody binding fragment thereof or an nucleic acidencoding such antibodies or antibody binding fragments thereof, forexample the antibody binding fragment can be one or more of the CDRsgrafted onto a protein scaffold. In some examples, the antibodies, or anantibody binding fragment thereof or an nucleic acid encoding suchantibodies or antibody binding fragments thereof, can be used inpost-exposure prophylaxis. In some examples, antibodies, or an antibodybinding fragment thereof or an nucleic acid encoding such antibodies orantibody binding fragments thereof, can be used to eliminate the viralreservoir. For example a therapeutically effective amount of theantibodies, or an antibody binding fragment thereof or an nucleic acidencoding such antibodies or antibody binding fragments thereof can beadministered to a subject being treated with anti-viral therapy. In someexamples the antibodies, or an antibody binding fragment thereof ismodified such that it is directly cytotoxic to infected cells, or usesnatural defenses such as complement, antibody dependent cellularcytotoxicity (ADCC), or phagocytosis by macrophages.

Methods to assay for neutralization activity include, but are notlimited to, a single-cycle infection assay as described in Martin et al.(2003) Nature Biotechnology 21:71-76. In this assay, the level of viralactivity is measured via a selectable marker whose activity isreflective of the amount of viable virus in the sample, and the IC50 isdetermined. In other assays, acute infection can be monitored in the PM1cell line or in primary cells (normal PBMC). In this assay, the level ofviral activity can be monitored by determining the p24 concentrationsusing ELISA. See, for example, Martin et al. (2003) Nature Biotechnology21:71-76.

HIV infection does not need to be completely eliminated for thecomposition to be effective. For example, a composition can decrease HIVinfection by a desired amount, for example by at least 10%, at least20%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 98%, or even at least 100% (elimination ofdetectable HIV infected cells), as compared to HIV infection in theabsence of the composition. In example, the cell is also contacted withan effective amount of an additional agent, such as anti-viral agent.The cell can be in vivo or in vitro. The methods can includeadministration of one on more additional agents known in the art. Inadditional examples, HIV replication can be reduced or inhibited bysimilar methods. HIV replication does not need to be completelyeliminated for the composition to be effective. For example, acomposition can decrease HIV replication by a desired amount, forexample by at least 10%, at least 20%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, oreven at least 100% (elimination of detectable HIV), as compared to HIVreplication in the absence of the composition. In one example, the cellis also contacted with an effective amount of an additional agent, suchas anti-viral agent. The cell can be in vivo or in vitro.

Compositions are provided that include one or more of the antibodiesthat specifically bind gp41, or an antibody binding fragment thereof oran nucleic acid encoding such antibodies or antibody binding fragmentsthereof, that are disclosed herein in a carrier. The compositions can beprepared in unit dosage forms for administration to a subject. Theamount and timing of administration are at the discretion of thetreating physician to achieve the desired purposes. The antibody can beformulated for systemic or local administration. In one example, theantibody that specifically binds gp41, or an antibody binding fragmentthereof or a nucleic acid encoding such antibodies or antibody bindingfragments thereof, is formulated for parenteral administration, such asintravenous administration.

The compositions for administration can include a solution of theantibody that specifically binds gp41, or an antibody binding fragmentthereof or an nucleic acid encoding such antibodies or antibody bindingfragments thereof, dissolved in a pharmaceutically acceptable carrier,such as an aqueous carrier. A variety of aqueous carriers can be used,for example, buffered saline and the like. These solutions are sterileand generally free of undesirable matter. These compositions may besterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example, sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like. The concentration ofantibody in these formulations can vary widely, and will be selectedprimarily based on fluid volumes, viscosities, body weight and the likein accordance with the particular mode of administration selected andthe subject's needs.

A typical pharmaceutical composition for intravenous administrationincludes about 0.1 to 10 mg of antibody per subject per day. Dosagesfrom 0.1 up to about 100 mg per subject per day may be used,particularly if the agent is administered to a secluded site and notinto the circulatory or lymph system, such as into a body cavity or intoa lumen of an organ. Actual methods for preparing administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa.(1995).

Antibodies, or an antibody binding fragment thereof or an nucleic acidencoding such antibodies or antibody binding fragments thereof, may beprovided in lyophilized form and rehydrated with sterile water beforeadministration, although they are also provided in sterile solutions ofknown concentration. The antibody solution, or an antibody bindingfragment thereof or an nucleic acid encoding such antibodies or antibodybinding fragments thereof, is then added to an infusion bag containing0.9% sodium chloride, USP, and typically administered at a dosage offrom 0.5 to 15 mg/kg of body weight. Considerable experience isavailable in the art in the administration of antibody drugs, which havebeen marketed in the U.S. since the approval of RITUXAN® in 1997.Antibodies, or an antibody binding fragment thereof or an nucleic acidencoding such antibodies or antibody binding fragments thereof, can beadministered by slow infusion, rather than in an intravenous push orbolus. In one example, a higher loading dose is administered, withsubsequent, maintenance doses being administered at a lower level. Forexample, an initial loading dose of 4 mg/kg may be infused over a periodof some 90 minutes, followed by weekly maintenance doses for 4-8 weeksof 2 mg/kg infused over a 30 minute period if the previous dose was welltolerated.

A therapeutically effective amount of a human gp41-specific antibody, oran antibody binding fragment thereof or an nucleic acid encoding suchantibodies or antibody binding fragments thereof, will depend upon theseverity of the disease and/or infection and the general state of thepatient's health. A therapeutically effective amount of the antibody isthat which provides either subjective relief of a symptom(s) or anobjectively identifiable improvement as noted by the clinician or otherqualified observer. These compositions can be administered inconjunction with another therapeutic agent, either simultaneously orsequentially.

In one embodiment, administration of the antibody, or an antibodybinding fragment thereof or an nucleic acid encoding such antibodies orantibody binding fragments thereof, results in a reduction in theestablishment of HIV infection and/or reducing subsequent HIV diseaseprogression in a subject. A reduction in the establishment of HIVinfection and/or a reduction in subsequent HIV disease progressionencompass any statistically significant reduction in HIV activity. Insome embodiments, methods are disclosed for treating a subject with anHIV-1 infection. These methods include administering to the subject atherapeutically effective amount of an antibody, or a nucleic acidencoding the antibody, thereby preventing or treating the HIV-1infection.

Studies have shown that the rate of HIV transmission from mother toinfant is reduced significantly when zidovudine is administered toHIV-infected women during pregnancy and delivery and to the offspringafter birth (Connor et al., 1994 Pediatr Infect Dis J 14: 536-541).Several studies of mother-to-infant transmission of HIV havedemonstrated a correlation between the maternal virus load at deliveryand risk of HIV transmission to the child. The present disclosureprovides isolated human monoclonal antibodies that are of use indecreasing HIV-transmission from mother to infant. Thus, in someexamples, a therapeutically effective amount of a human gp41-specificantibody, or an antibody binding fragment thereof or an nucleic acidencoding such antibodies or antibody binding fragments thereof, isadministered in order to prevent transmission of HIV, or decrease therisk of transmission of HIV, from a mother to an infant. In someexamples, a therapeutically effective amount of the antibody, or anantibody binding fragment thereof or an nucleic acid encoding suchantibodies or antibody binding fragments thereof, is administered tomother and/or to the child at childbirth. In other examples, atherapeutically effective amount of the antibody is administered to themother and/or infant prior to breast feeding in order to prevent viraltransmission to the infant or decrease the risk of viral transmission tothe infant. In some embodiments, both a therapeutically effective amountof the antibody and a therapeutically effective amount of another agent,such as zidovudine, is administered to the mother and/or infant.

For any application, the antibody, or an antibody binding fragmentthereof or an nucleic acid encoding such antibodies or antibody bindingfragments thereof, can be combined with anti-retroviral therapy.Anti-retroviral drugs are broadly classified by the phase of theretrovirus life-cycle that the drug inhibits. The disclosed antibodiescan be administered in conjunction with nucleoside analogreverse-transcriptase inhibitors (such as zidovudine, didanosine,zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir,and apricitabine), nucleotide reverse transcriptase inhibitors (such astenofovir and adefovir), non-nucleoside reverse transcriptase inhibitors(such as efavirenz, nevirapine, delavirdine, etravirine, andrilpivirine), protease inhibitors (such as saquinavir, ritonavir,indinavir, nelfinavir, amprenavir, lopinavir, fosamprenavir, atazanavir,tipranavir, and darunavir), entry or fusion inhibitors (such asmaraviroc and enfuvirtide), maturation inhibitors, (such as bevirimatand vivecon), or a broad spectrum inhibitors, such as naturalantivirals. In some examples, a disclosed antibody or active fragmentthereof or nucleic acids encoding such is administered in conjunctionwith IL-15, or conjugated to IL-15.

In some examples, a subject is further administered one or moreadditional antibodies that bind HIV glycoproteins, such as gp120 andgp41. Examples of neutralizing antibodies that can be administered inconjunction with the disclosed antibodies can be found in InternationalPatent Publication No. WO 2011/038290, published Mar. 31, 2011, which isspecifically incorporated herein by reference in its entirety.

Single or multiple administrations of the compositions including theantibodies disclosed herein are administered depending on the dosage andfrequency as required and tolerated by the patient. In any event, thecomposition should provide a sufficient quantity of at least one of theantibodies disclosed herein to effectively treat the patient. The dosagecan be administered once, but may be applied periodically until either atherapeutic result is achieved or until side effects warrantdiscontinuation of therapy. In one example, a dose of the antibody isinfused for thirty minutes every other day. In this example, about oneto about ten doses can be administered, such as three or six doses canbe administered every other day. In a further example, a continuousinfusion is administered for about five to about ten days. The subjectcan be treated at regular intervals, such as monthly, until a desiredtherapeutic result is achieved. Generally, the dose is sufficient totreat or ameliorate symptoms or signs of disease without producingunacceptable toxicity to the patient.

Controlled-release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995). Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein, suchas a cytotoxin or a drug, as a central core. In microspheres thetherapeutic is dispersed throughout the particle. Particles,microspheres, and microcapsules smaller than about 1 μm are generallyreferred to as nanoparticles, nanospheres, and nanocapsules,respectively. Capillaries have a diameter of approximately 5 μm so thatonly nanoparticles are administered intravenously. Microparticles aretypically around 100 μm in diameter and are administered subcutaneouslyor intramuscularly. See, for example, Kreuter, J., Colloidal DrugDelivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y.,pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled DrugDelivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp.315-339, (1992).

Polymers can be used for ion-controlled release of the antibodycompositions disclosed herein. Various degradable and nondegradablepolymeric matrices for use in controlled drug delivery are known in theart (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, theblock copolymer, polaxamer 407, exists as a viscous yet mobile liquid atlow temperatures but forms a semisolid gel at body temperature. It hasbeen shown to be an effective vehicle for formulation and sustaineddelivery of recombinant interleukin-2 and urease (Johnston et al.,Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech.44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa. (1993)). Numerous additionalsystems for controlled delivery of therapeutic proteins are known (seeU.S. Pat. Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028;4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164;5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).

In some examples, a subject is administered the DNA encoding theantibody or antibody binding fragments thereof, for example the antibodybinding fragment can be one or more of the CDRs grafted onto a proteinscaffold, to provide in vivo antibody production, for example using thecellular machinery of the subject. Immunization by nucleic acidconstructs is well known in the art and taught, for example, in U.S.Pat. Nos. 5,643,578, and 5,593,972 and 5,817,637. 5,880,103 describesseveral methods of delivery of nucleic acids encoding to an organism.The methods include liposomal delivery of the nucleic acids. Suchmethods can be applied to the production of an antibody, or antibodybinding fragments thereof, by one of ordinary skill in the art.

One approach to administration of nucleic acids is direct administrationwith plasmid DNA, such as with a mammalian expression plasmid. Thenucleotide sequence encoding the disclosed antibody, or antibody bindingfragments thereof, can be placed under the control of a promoter toincrease expression.

In another approach to using nucleic acids, a disclosed antibody, orantibody binding fragments thereof can also be expressed by attenuatedviral hosts or vectors or bacterial vectors. Recombinant vaccinia virus,adeno-associated virus (AAV), herpes virus, retrovirus, cytomegalovirusor other viral vectors can be used to express the antibody. For example,vaccinia vectors and methods useful protocols are described in U.S. Pat.No. 4,722,848. BCG (Bacillus Calmette Guerin) provides another vectorfor expression of the disclosed antibodies (see Stover, Nature351:456-460, 1991).

In one embodiment, a nucleic acid encoding a disclosed antibody, orantibody binding fragments thereof, is introduced directly into cells.For example, the nucleic acid can be loaded onto gold microspheres bystandard methods and introduced into the skin by a device such asBio-Rad's HELIOS™ Gene Gun. The nucleic acids can be “naked,” consistingof plasmids under control of a strong promoter.

Typically, the DNA is injected into muscle, although it can also beinjected directly into other sites. Dosages for injection are usuallyaround 0.5 μg/kg to about 50 mg/kg, and typically are about 0.005 mg/kgto about 5 mg/kg (see, e.g., U.S. Pat. No. 5,589,466).

D. Diagnostic Methods and Kits

A method is provided herein for the detection of the expression of gp41in vitro or in vivo. In one example, expression of gp41 is detected in abiological sample, and can be used to detect HIV-1 infection as thepresence of HIV-1 in a sample. The sample can be any sample, including,but not limited to, tissue from biopsies, autopsies and pathologyspecimens. Biological samples also include sections of tissues, forexample, frozen sections taken for histological purposes. Biologicalsamples further include body fluids, such as blood, serum, plasma,sputum, spinal fluid or urine.

In several embodiments, a method is provided for detecting AIDS and/oran HIV-1 infection in a subject. The disclosure provides a method fordetecting HIV-1 in a biological sample, wherein the method includescontacting a biological sample with the antibody under conditionsconducive to the formation of an immune complex, and detecting theimmune complex, to detect the gp41 in the biological sample. In oneexample, the detection of gp41 in the sample indicates that the subjecthas an HIV infection. In another example, the detection of gp41 in thesample indicates that the subject has AIDS. In another example,detection of gp41 in the sample confirms a diagnosis of AIDS and/or anHIV-1 infection in a subject.

In some embodiments, the disclosed antibodies are used to test vaccines.For example to test if a vaccine composition assumes the sameconformation as a gp41 peptide. Thus provided herein is a method fortesting a vaccine, wherein the method includes contacting a samplecontaining the vaccine, such as a gp41 immunogen, with the antibodyunder conditions conducive to the formation of an immune complex, anddetecting the immune complex, to detect the vaccine in the sample. Inone example, the detection of the immune complex in the sample indicatesthat vaccine component, such as such as a gp41 immunogen assumes aconformation capable of binding the antibody.

In one embodiment, the antibody is directly labeled with a detectablelabel. In another embodiment, the antibody that binds gp41 (the firstantibody) is unlabeled and a second antibody or other molecule that canbind the antibody that binds gp41 is utilized. As is well known to oneof skill in the art, a second antibody is chosen that is able tospecifically bind the specific species and class of the first antibody.For example, if the first antibody is a human IgG, then the secondaryantibody may be an anti-human-IgG. Other molecules that can bind toantibodies include, without limitation, Protein A and Protein G, both ofwhich are available commercially.

Suitable labels for the antibody or secondary antibody are describedabove, and include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, magnetic agents and radioactivematerials. Non-limiting examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase. Non-limiting examples of suitable prosthetic groupcomplexes include streptavidin/biotin and avidin/biotin. Non-limitingexamples of suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Anon-limiting exemplary luminescent material is luminol; a non-limitingexemplary a magnetic agent is gadolinium, and non-limiting exemplaryradioactive labels include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

The immunoassays and methods disclosed herein can be used for a numberof purposes. Kits for detecting a polypeptide will typically include anantibody that binds gp41, such as any of the antibodies disclosedherein. In some embodiments, an antibody fragment, such as an Fvfragment or a Fab is included in the kit. In a further embodiment, theantibody is labeled (for example, with a fluorescent, radioactive, or anenzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use. The instructional materials may be written, in anelectronic form (such as a computer diskette or compact disk) or may bevisual (such as video files). The kits may also include additionalcomponents to facilitate the particular application for which the kit isdesigned. Thus, for example, the kit may additionally contain means ofdetecting a label (such as enzyme substrates for enzymatic labels,filter sets to detect fluorescent labels, appropriate secondary labelssuch as a secondary antibody, or the like). The kits may additionallyinclude buffers and other reagents routinely used for the practice of aparticular method. Such kits and appropriate contents are well known tothose of skill in the art.

In one embodiment, the diagnostic kit includes an immunoassay. Althoughthe details of the immunoassays may vary with the particular formatemployed, the method of detecting gp41 in a biological sample generallyincludes the steps of contacting the biological sample with an antibodywhich specifically reacts, under immunologically reactive conditions, togp41. The antibody is allowed to specifically bind under immunologicallyreactive conditions to form an immune complex, and the presence of theimmune complex (bound antibody) is detected directly or indirectly.

E. Methods of Identifying Antibodies of Interest

Methods are provided for producing a monoclonal antibody thatspecifically binds to a target antigen. These methods include isolatinga population of memory B cells from a subject that has been exposed tothe target antigen, wherein the memory B cells are CD19+IgA-IgD-IgM- Bcells. The population of the isolated memory B cells is contacted withan effective amount of IL-21, IL-2 and CD40 ligand (CD40L), and mRNA isisolated from the population of isolated memory B cells. Nucleic acidsencoding the variable heavy chains and the variable light chains ofantibodies are isolated from cells, and the variable heavy chains andthe variable light chains are expressed. A monoclonal antibody includinga variable heavy chain and a variable light chain that specificallybinds to the target antigen is then selected from combinations of thevariable heavy chains and the variable light chains.

In some embodiments, a population of memory B cells is isolated from abiological sample from a subject that has been previously exposed to theantigen of interest. The population of memory B cells is divided intosub-populations which are contacted with an effective amount of CD40L,IL-2 and IL-21 for a sufficient amount of time for the memory B cells toundergo cell division and produce antibodies. The presence or absence ofantibodies that specifically binds to the antigen of interest isdetermined for the subpopulations of memory B cells. If it is determinedthat a subpopulation of memory B cells produces antibodies thatspecifically bind to the antigen of interest, then that subpopulation isselected. The nucleic acid sequence encoding the heavy and light chainvariable domains of the antibodies produced by memory B cells of theselected subpopulation can be determined, and monoclonal antibodiescontaining the heavy and light chain variable regions of antibodiesproduced by the selected subpopulation of memory B cells produced. Themonoclonal antibodies are assayed for specific binding to the antigen oninterest, and antibodies that specifically bind to the antigen ofinterest are selected, thereby identifying an antibody that specificallybinds to the antigen of interest.

Methods are also provided for isolating the repertoire of B cellsspecific for a target antigen from a subject. These methods includeisolating a population of memory B cells from a subject that has beenexposed to the target antigen, wherein the memory B cells areCD19+IgA-IgD-IgM- B cells. The population of the isolated memory B cellsis contacted with an effective amount of IL-21, IL-2 and CD40, and Bcells are selected from the population that expresses antibodies thatspecifically bind the target antigen. These methods can also includeisolating the library of nucleic acids encoding the variable heavychains and the variable light chains of immunoglobulins are isolatedfrom nucleic acids. The library of variable heavy chains and thevariable light chains are then expressed to isolate the repertoire of Bcells specific for the target antigen from the subject.

A humoral repertoire, including but not limited to the full humoralrepertoire, to an entity, such as a pathogen or vaccine, can providemulti-dimensional information (e.g. specificities, affinities,stabilities, gene segment sequence preferences, etc) that could beconsidered a “profile” of a subject's humoral response. Quantitation ofthese parameters (Story et al., 2008 PNAS 105(46):17902-17907) can beused to correlate with protection from a pathogen or failure to protect.This information could then inform vaccine design in an iterativefashion, provide the basis for a multi-parameter diagnostic assay forspecific antigens, or be directly used to identify single or multipleneutralizing antibodies against a given pathogen.

In some embodiments, the antibodies can be characterized. For example,multiparametric datasets can be collected that describe thecharacteristics, e.g. specificities, affinities, stabilities, isotypes,gene segment sequence preferences, etc. (Story et al., 2008 PNAS105(46):17902-17907. In some representative, non-limiting embodiments,the profile or multiparametric dataset can be used to inform vaccinedesign in an iterative fashion, provide the basis for a multi-parameterdiagnostic assay for specific antigens, or be directly used to identifysingle or multiple neutralizing antibodies against a given pathogen.

Thus, the methods disclosed herein can be used to isolate one or moremonoclonal antibodies that specifically bind a target antigen, and/orcan be used to isolate the B cell repertoire that bind the targetantigen in a sample or in a subject. The target antigen can be from apathogen, including viruses, parasites, fungi and bacteria. In someembodiments, pathogen is a virus, such as, but not limited to a virusfrom one of the following families: Retroviridae (for example, humanimmunodeficiency virus (HIV); human T-cell leukemia viruses (HTLV);Picornaviridae (for example, polio virus, hepatitis A virus; hepatitis Cvirus; enteroviruses, human coxsackie viruses, rhinoviruses,echoviruses; foot-and-mouth disease virus); Calciviridae (such asstrains that cause gastroenteritis); Togaviridae (for example, equineencephalitis viruses, rubella viruses); Flaviridae (for example, dengueviruses; yellow fever viruses; West Nile virus; St. Louis encephalitisvirus; Japanese encephalitis virus; and other encephalitis viruses);Coronaviridae (for example, coronaviruses; severe acute respiratorysyndrome (SARS) virus; Rhabdoviridae (for example, vesicular stomatitisviruses, rabies viruses); Filoviridae (for example, Ebola viruses);Paramyxoviridae (for example, parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus (RSV)); Orthomyxoviridae (forexample, influenza viruses); Bunyaviridae (for example, Hantaan viruses;Sin Nombre virus, Rift Valley fever virus; bunya viruses, phlebovirusesand Nairo viruses); Arena viridae (hemorrhagic fever viruses; Machupovirus; Junin virus); Reoviridae (e.g., reoviruses, orbiviurses androtaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyomaviruses; BK-virus); Adenoviridae (most adenoviruses); Herpesviridae(herpes simplex virus (HSV)-1 and HSV-2; cytomegalovirus (CMV);Epstein-Barr virus (EBV); varicella zoster virus (VZV); and other herpesviruses, including HSV-6); Poxyiridae (variola viruses, vacciniaviruses, pox viruses); and Iridoviridae (such as African swine fevervirus); Filoviridae (for example, Ebola virus; Marburg virus);Caliciviridae (for example, Norwalk viruses) and unclassified viruses(for example, the etiological agents of Spongiform encephalopathies, theagent of delta hepatitis (thought to be a defective satellite ofhepatitis B virus); and astroviruses).

In other embodiments, the target antigen is an antigen from a bacteria,such as, but not limited to, Helicobacter pyloris, Borelia burgdorferi,Legionella pneumophilia, Mycobacteria sps (such as. M. tuberculosis, M.avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcusaureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeriamonocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusanthracia, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Bacteroides sp., Fusobacterium nucleaturn, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira, orActinomyces israelli.

In further embodiments, the antigen is from a fungus, such asCryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis,Blastomyces dermatitidis, Chlamydia trachomatis, or Candida albicans. Inother embodiments, antigen is from a parasite, such as, but not limitedto, Plasmodium falciparum or Toxoplasma gondii.

In some embodiments, the antigen is a cancer antigen. The cancer can bea solid tumor or a hematogenous cancer. In particular examples, thesolid tumor is a sarcoma or a carcinoma, such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, or anothersarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testiculartumor, bladder carcinoma, or a CNS tumor (such as a glioma, astrocytoma,medulloblastoma, craniopharyogioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma or retinoblastoma).

In some examples, the hematogenous cancer is a leukemia, such as anacute leukemia (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia); a chronic leukemia (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia ormyelodysplasia.

Tumor antigens are well known in the art and include, for example,carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG),alpha-fetoprotein (AFP), lectin-reactive AFP, (AFP-L3), thyroglobulin,RAGE-1, MN-CA IX, human telomerase reverse transcriptase (hTERT), RU1,RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein,PSMA, Her2/neu, survivin and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), melanoma-associated antigen (MAGE), ELF2M,neutrophil elastase, ephrinB2 and CD22. The CH2 or CH3 domain moleculescan also bind any cancer-related proteins, such IGF-I, IGF-II, IGR-IR ormesothelin. Additional tumor associated antigens are provided in theTable below:

Exemplary tumors and their tumor antigens Tumor Tumor Associated TargetAntigens Acute myelogenous leukemia Wilms tumor 1 (WT1), preferentiallyexpressed antigen of melanoma (PRAME), PR1, proteinase 3, elastase,cathepsin G Chronic myelogenous leukemia WT1, PRAME, PR1, proteinase 3,elastase, cathepsin G Myelodysplastic syndrome WT1, PRAME, PR1,proteinase 3, elastase, cathepsin G Acute lymphoblastic leukemia PRAMEChronic lymphocytic leukemia Survivin Non-Hodgkin's lymphoma SurvivinMultiple myeloma New York esophagus 1 (NY-Eso1) Malignant melanoma MAGE,MART, Tyrosinase, PRAME GP100 Breast cancer WT1, Herceptin Lung cancerWT1 Prostate cancer Prostate-specific antigen (PSA) Colon cancerCarcinoembryonic antigen (CEA) Renal cell carcinoma (RCC) Fibroblastgrowth factor 5 (FGF-5)

In some embodiments, the antigen is a self antigen. The antigen can bean antigen associated with an autoimmune disease, such as rheumatoidarthritis, juvenile oligoarthritis, collagen-induced arthritis,adjuvant-induced arthritis, Sjögren's syndrome, multiple sclerosis,experimental autoimmune encephalomyelitis, inflammatory bowel disease(for example, Crohn's disease, ulcerative colitis), autoimmune gastricatrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes,non-obese diabetes, myasthenia gravis, Grave's disease, Hashimoto'sthyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemiclupus erythematosis, autoimmune thrombocytopenia purpura, Goodpasture'ssyndrome, Addison's disease, systemic sclerosis, polymyositis,dermatomyositis, autoimmune hemolytic anemia or pernicious anemia.

Isolating B Cells

In several embodiments, a population of cells including memory B cellsis obtained from a subject. Typically, a substantially pure populationof memory B cells (such as CD19⁺IgA⁻, IgD⁻, IgM⁻ B cells) are isolated.Typically, the isolated population of cells is enriched for memory Bcells.

The population of cells including memory B cells can be isolated from abiological sample obtained from a subject of interest. Exemplarybiological samples for use with the present methods include bone marrow,spleen, lymph node, blood, e.g., peripheral blood. However, thebiological sample can also include any other source from which memory Bcells can be isolated, including: tissue biopsy, surgical specimens,fine needle aspirates, autopsy material, and the like. In severalembodiments, the biological sample is obtained from a subject that hasbeen exposed to an antigen of interest. The subject may be any animal,preferably a mammal or a human. The subject may have a disease or acondition including a tumor, an infectious disease, or an autoimmunedisease, or have been immunized. In certain aspects, the subject mayrecover or survive from a disease or a condition such as a tumor, aninfectious disease, or an autoimmune disease. In further aspects, thesubject may be under or after prevention and treatment for a disease ora condition, such as cancer therapy or infection disease therapy, orvaccination. For example, the subject has or has been exposed to anantigen which is an infectious agent, a tumor antigen, a tumor cell, anallergen or a self-antigen. Such an infectious agent may be anypathogenic viruses, pathogenic bacteria, fungi, protozoa, multicellularparasites, and aberrant proteins such as prions, as wells as nucleicacids or antigens derived therefrom. An allergen could be anynonparasitic antigen capable of stimulating a type-I hypersensitivityreaction in individuals, such as many common environmental antigens.

Fluorescence activated cell sorting (FACS) can be used to sort (isolate)cells, such as populations of memory B cells, by contacting the cellswith an appropriately labeled antibody and sorting the cells based onbinding of the labeled antibody to the cell. In one embodiment, severalantibodies (such as antibodies that bind CD19, IgA, IgD, and/or IgM) andFACS sorting can be used to produce substantially purified populationsof memory B cells. These methods are known in the art, and exemplaryprotocols are described herein.

FACS employs a plurality of color channels, low angle and obtuselight-scattering detection channels, and impedance channels, among othermore sophisticated levels of detection, to separate or sort cellslabeled or not labeled with a detectable marker. Any FACS technique canbe employed as long as it is not detrimental to the viability of thedesired cells. (For exemplary methods of FACS see U.S. Pat. No.5,061,620). In one example, a FACSARIA III® cell sorter (BectonDickinson, Franklin Lakes, N.J.) is used. Antibodies can be conjugatedto biotin, which then can be removed with avidin or streptavidin boundto a support, or fluorochromes, which can be used with a FACS, to enablecell separation.

However, other techniques of differing efficacy can be employed topurify and isolate desired populations of cells. The separationtechniques employed should maximize the retention of viability of thefraction of the cells to be collected. The particular technique employedwill, of course, depend upon the efficiency of separation, cytotoxicityof the method, the ease and speed of separation, and what equipmentand/or technical skill is required.

Separation procedures include magnetic separation, using antibody-coatedmagnetic beads, affinity chromatography, cytotoxic agents, either joinedto a monoclonal antibody or used in conjunction with complement, and“panning,” which utilizes a monoclonal antibody attached to a solidmatrix, or another convenient technique. Antibodies attached to magneticbeads and other solid matrices, such as agarose beads, polystyrenebeads, hollow fiber membranes and plastic petri dishes, allow for directseparation. Cells that are bound by the antibody can be removed from thecell suspension by simply physically separating the solid support fromthe cell suspension. The exact conditions and duration of incubation ofthe cells with the solid phase-linked antibodies will depend uponseveral factors specific to the system employed. The selection ofappropriate conditions, however, is well within the skill in the art.

The unbound cells then can be collected (when negative selection forbinding to the immunobeads) is being used) or washed away withphysiologic buffer (when positive selection is benign used for bindingto the immunobeads) after sufficient time has been allowed for the cellsexpressing a marker of interest (e.g., CD19) to bind to the solid-phaselinked antibodies. The bound cells are then separated from the solidphase by any appropriate method, depending mainly upon the nature of thesolid phase and the antibody employed. After a first round of selectionusing magnet beads, a second round (and additional rounds) can be usedto further isolate a cell population of interest.

In some embodiments, cells expressing CD19 are separated from othercells by positive selection for the cell-surface expression of CD19. Inone specific, non-limiting example, CD19+ cells are positively selectedusing FACS by labeling CD19⁺ cells with a CD19 specific antibodyconjugated to a detectable marker, and then using FACS to select cellslabeled with the antibody conjugated to the detectable marker. CD19specific antibodies conjugated to detectable markers are known and arecommercially available, for example from BD Bioscience, Franklin Lakes,N.J. In another specific, non-limiting example, CD19⁺ cells arepositively selected by magnetic bead separation, wherein magnetic beadsare coated with CD19 reactive monoclonal antibodies, and cell that arecaptured by the CD19 reactive immunobeads are collected. The CD19⁺ cellsare then removed from the magnetic beads.

In other embodiments, cells that do not express IgA on the cell surfaceare separated from other cells by the lack of cell-surface expression ofIgA. In one specific, non-limiting example, IgA⁻ cells are negativelyselected using FACS by labeling IgA⁺ cells with an IgA specific antibodyconjugated to a detectable marker, and then using FACS to select cellsthat are not labeled with the IgA specific antibody conjugated to thedetectable marker. IgA specific antibodies conjugated to detectablemarkers are known and are commercially available, for example fromJackson ImmunoResearch Laboratories, Inc. West Grove, Pa. In anotherspecific, non-limiting example, IgA⁻ cells are negatively selected bymagnetic bead separation, wherein magnetic beads are coated with IgAreactive monoclonal antibody and cells that are not captured by theimmunobeads are collected.

In other embodiments, cells that do not express IgD on the cell surfaceare separated from other cells by the lack of cell-surface expression ofIgD. In one specific, non-limiting example, IgD⁻ cells are negativelyselected using FACS by labeling IgD⁺ cells with an IgD specific antibodyconjugated to a detectable marker, and then using FACS to select cellsthat are not labeled with the IgD specific antibody conjugated to thedetectable marker. IgD specific antibodies conjugated to detectablemarkers are known and are commercially available, for example from BDPharmingen, Franklin Lakes, N.J. In another specific, non-limitingexample, IgD⁻ cells are negatively selected by magnetic bead separation,wherein magnetic beads are coated with IgD reactive monoclonal antibodyand cells that are not captured by the immunobeads are collected.

In other embodiments, cells that do not express IgM on the cell surfaceare separated from other cells by the lack of cell-surface expression ofIgM. In one specific, non-limiting example, IgM⁻ cells are negativelyselected using FACS by labeling IgM⁺ cells with an IgM specific antibodyconjugated to a detectable marker, and then using FACS to select cellsthat are not labeled with the IgM specific antibody conjugated to thedetectable marker. IgM specific antibodies conjugated to detectablemarkers are known and are commercially available, for example fromJackson ImmunoResearch Laboratories, Inc. West Grove, Pa. In anotherspecific, non-limiting example, IgM⁻ cells are negatively selected bymagnetic bead separation, wherein magnetic beads are coated with IgMreactive monoclonal antibody and cells that are not captured by theimmunobeads are collected.

In further embodiments, cells that express CD19, but do not express IgA,IgD or IgM on the cell surface are separated from other cells bypositively selecting for CD19 cell surface expression and negativelyselecting for IgA, IgD and IgM expression on the cell surface. Usingsuch methods, CD19⁺IgA⁻IgD⁻IgM⁻ cells can be collected. In one specific,non-limiting example, CD19⁺IgA⁻IgD⁻IgM⁻ cells are selected using FACS bylabeling cells with four antibodies specific to CD19, IgA, IgD, and IgM,each of which is conjugated to a detectable marker that can bedifferentially detected using FACS analysis. FACS is then used to sortCD19⁺IgA⁻IgD⁻IgM⁻ cells. One of skill in the art can readily use FACSand set appropriate gates to isolate CD19⁺IgA⁻IgD⁻IgM⁻ cells.

Contacting B Cells with CD40L, IL-2 and IL-21

In several embodiments, isolated memory B cells are contacted withCD40L, IL-2 and IL-21 for a sufficient amount of time for the memory Bcells to undergo cell division and produce antibodies. In some apopulation of isolated memory B cells is contacted with CD40L, IL-2 andIL-21 by incubating the isolated population of memory B cells withCD40L, IL-2 and IL-21 for about 10 to about 15 days. In additionalembodiments, the isolated population of memory B cells is incubated withCD40L, IL-2 and IL-21 for about 13 days. In several embodiments, the Bcells are contacted with the CD40L, IL-2 and IL-21 for a sufficientamount of time for the memory B cells to undergo cell division andproduce antibodies. In this context, a sufficient amount of time can beat least 5 days, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21 or 22 days, for example from 5-22, 5-21, 10-20, 10-15, 11-16,or 13-15 days. The B cells can be contacted with the CD40L, IL-2 andIL-21 in the presence of growth media, such as Iscove's ModifiedDulbecco's Medium, (IMDM) with 10% Fetal Bovine Serum, or other tissueculture growth media for use in culturing B cells. The person ofordinary skill in the art is familiar with such media.

CD40L (also known as CD40 ligand or CD154) is a protein that is primaryexpressed, for example, on activated T cells, but is also found insoluble form. CD40L binds to CD40 on the cell surface of B cells, abinding event that can result in the activation of B cells,differentiation of mature B cells into plasma cells and memory cells,and production of antibodies. The person of ordinary skill in the art isfamiliar with CD40L, and CD40L is available commercially (see, forexample, Life Technologies Cat. No. PHP0024). In some embodiments, Bcells are contacted with CD40L by culturing the B cells with a cell linethat expresses CD40L, such as a CD40L feeder cell line. CD40L feedercell lines are known in the art (see, for example, Kershaw et al.,Cancer Res., 61: 7920-7924, 2001). Exemplary concentrations of CD40L foruse in the disclosed methods include 1-2000 international units permilliliter, such as 100 international units per milliliter. Exemplarynucleic acid and polypeptide sequences for human CD40L are available atthe NCBI website as GENBANK® Accession No. NM_000074.2 and GENBANK®Accession No. NP_000065.1, respectively, (as available on Jun. 13, 2012)which are incorporated herein by reference.

IL-2 is available commercially (see, for example, Life Technologies,Grand Island, N.Y., Cat. No. PHP0021). Exemplary nucleic acid andpolypeptide sequences for human IL-2 are available at the NCBI websiteas GENBANK® Accession No. NM_000586.3 and GENBANK® Accession No.NP_000577.2, respectively (as available on Jun. 13, 2012) which areincorporated herein by reference. Exemplary concentrations of IL-2 foruse in the disclosed methods include 10-200 international units permilliliter, such as 100 international units per milliliter.

IL-21 is also available commercially (see, for example, LifeTechnologies, Grand Island, N.Y., Cat. No. PHC0211). Exemplary nucleicacid and polypeptide sequences for human IL-21 are available at the NCBIwebsite as GENBANK® Accession No. NM021803 and GENBANK® Accession No.AF254069, respectively. These GENBANK® entries are incorporated byreference herein. In several embodiments, the isolated B cells arecontacted with about 10-100 ng/ml IL-21, such as about 10, 20, 30, 40,50, 60, 70, 80, 90, or 100 ng/ml IL-21, for example the memory B cellscan be contacted with from 10-100, 20-90, 30-80, 40-70, 40-60 or 45-55ng/ml IL-21. Exemplary concentrations of IL-2 for use in the disclosedmethods include 5-500 ng/ml IL-21, such as 50 ng/ml IL-21.

In several embodiments, contacting the memory B cells with thecombination of CD40L, IL-2 and IL21 provides synergy, that is, theamount of cell division and antibody production observed by the memory Bcells contacted with these three molecules is greater in combinationthen than the sum of the effect that results from using the moleculesseparately.

Selecting B Cells that Produce the Antibody of Interest

In some embodiments, the subpopulations of B cells that have beencontacted with CD40L, IL-2 and IL-21 for a period of time sufficient forthe memory B cells to undergo cell division and produce antibodies areassayed for expression of antibodies that specifically bind to theantigen of interest. Methods for determining if an antibody binds to anantigen of interest are familiar to the person of ordinary skill in theart, and include ELISA and neutralization assays.

Three representative general classes of screening methods that can beemployed (a) antibody capture assays; (b) antigen capture assays; and(c) functional screens. Combinations can also be employed. In antibodycapture assays, the antigen can be bound to a solid phase, monoclonalantibodies to be tested are allowed to bind to the antigen, unboundantibodies are removed by washing, and then the bound antibodies aredetected, e.g. by a secondary reagent such as a labeled antibody thatspecifically recognizes the antibody. For an antigen capture assay, theantigen can be labeled directly. In one embodiment, monoclonalantibodies to be tested can be bound to a solid phase and then reactedwith the optionally labeled antigen. Alternatively, the antibody-antigencomplex can be allowed to form by immunoprecipitation prior to bindingof the monoclonal antibody to be tested to a solid phase. Once theantibody-antigen complexes are bound to the solid phase, unbound antigencan be removed by washing and positives can be identified by detectingthe antigen.

Various functional screens exist for identifying monoclonal antibodieswith desired activities. Examples include a virus neutralization assay;the agonistic activity assay and blocking assay; keratinocyte monolayeradhesion assay and the mixed lymphocyte response (MLR) assay (Werther etal. J. Immunol. 157:4986-4995 (1996)); tumor cell growth inhibitionassays (as described in PCT Publication No. WO 89/06692, for example);antibody-dependent cellular-1-cytotoxicity (ADCC) andcomplement-mediated cytotoxicity (CDC) assays (U.S. Pat. No. 5,500,362);and hematopoiesis assays (see WO 95/27062). The class/subclass of theantibodies can be determined, e.g., by double-diffusion assays; antibodycapture on antigen-coated plates; and/or antibody capture on anti-IgGantibodies.

To screen for antibodies which bind to a particular epitope on theantigen of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping, e.g., as described in Champe et al. (J.Biol. Chem. 270:1388-1394 (1995)) can be performed to determine whetherthe antibody binds an epitope of interest.

If it is determined that a subpopulation of memory B cells producesantibodies that specifically bind to the antigen of interest, thenadditional steps can be taken to isolate the a monoclonal antibody thatspecifically binds to the antigen of interest from the subpopulation.For example, the variable region of the heavy chain and the light chainof the immunoglobulin genes from the B cells in the subpopulation areisolated, monoclonal antibodies containing the variable region of theheavy chain and the light chains are generating, and the specificbinding acidity of the monoclonal antibodies for the antigen of interestis assay (for example, by ELISA or neutralization assay).

In certain aspects, sequence information of nucleic acid sequences canbe obtained. Obtaining the nucleic acid sequence information may includedetermining the nucleic acid sequence. For determining the nucleic acidsequences, any nucleic acid sequencing methods known in the art may beused, including high throughput DNA sequencing. Non-limiting examples ofhigh-throughput sequencing methods include sequencing-by-synthesis(e.g., 454 sequencing), sequencing-by-ligation,sequencing-by-hybridization, single molecule DNA sequencing, multiplexpolony sequencing, nanopore sequencing, or a combination thereof.

In a further embodiment the method can include (a) isolating RNA from asub-population of B cells; (b) transcribing said RNA to cDNA; (c)amplifying from said cDNA said first pool of DNA molecules using a firstmixture of oligonucleotides including at least two oligonucleotidescapable of amplifying heavy chain variable domain coding regions; (d)amplifying from said cDNA said second pool of DNA molecules using asecond mixture of oligonucleotides including at least twooligonucleotides capable of amplifying light chain variable domaincoding regions; and optionally (e) linking specimens of said first andsaid second pool of DNA molecules to each other by a DNA encoding saidlinker region (LR).

The cloning of variable regions is a standard procedure generally knownin the art and has been described for various species, including humans,non-human primates, mouse, rabbit, and chicken. For review see BarbasIII et al. (eds.), Phage Display—A Laboratory manual, Cold SpringHarbour Press, 2001, in particular the chapter Andris-Widhopf et al.,Generation of Antibody Libraries: PCR Amplification and Assembly ofLight- and Heavy-chain Coding Sequences, therein. Andris-Widhopf et al.discloses sequences of oligonucleotides capable of amplifying variableregion coding regions (VR coding regions), preferably have chainvariable domain coding regions or light chain variable domain codingregions. Furthermore, oligonucleotides capable of amplifying heavy chainvariable domain coding regions or light chain variable domain codingregions, preferably human heavy chain variable domain coding regions orlight chain variable domain coding regions, can be designed by theartisan by comparing known sequences of antibody coding regions whichare available from databases such as, for example, Immunogenetics(imgt.cines.fr/), Kabat (Kabatdatabase.com), and Vbase(vbase.mrc-cpe.cam.ac.uk/), and by identifying consensus sequencessuitable for primer design. Oligonucleotides capable of amplifying heavychain variable domain coding regions or light chain variable domaincoding regions, wherein the primers can include suitable restrictionsites for the cloning of the amplified products and wherein theoligonucleotides also encode a linker region are known in the art.Additional strategies for amplifying and cloning variable domains aredescribed in Sblattero and Bradbury (1998) Immunotechnology 3:271-278and Weitkamp et al. (2003), J. Immunol. Meth. 275:223-237.

In some examples, the variable region of the heavy chain and the lightchain of the immunoglobulin genes can be amplified by RT-PCR using knownmethods (see, e.g., Tiller et al., J. Immunological Methods,329:112-124, 2008). PCR product including the V_(H) or V_(L)-region DNAcan be cloned into corresponding Igγ1, Igκ and Igλ expression vectors,which can be used to co-transfected into a permissive cell line (such as293T cells) for expression and production of monoclonal antibody. Insome examples, full-length IgG1 can be purified using standardprocedures, and then tested for binding to the antigen of interest (forexample using ELISA or neutralization assay). The person of ordinaryskill in the art will understand that the expression vectors can beexpressed in any permissive cell line or subject for testing (forexample in a mammalian cell line, a plant cell line, or using a viralexpression vector for expression in a cell line or organism. Further,proteins including the sequence encoded by the V_(H) or V_(L)-region DNAcan be produced synthetically for testing.

Several Embodiments Concerning Methods of Identifying Antibodies ofInterest

Several embodiments include a method for producing a monoclonal antibodythat specifically binds to a target antigen, wherein the methodincludes: (a) isolating a population of memory B cells from a subjectthat has been exposed to the target antigen, wherein the memory B cellsare CD19+IgA-IgD-IgM- B cells; (b) contacting the isolated population ofmemory B cells with an effective amount of IL-21, IL-2 and CD40; (c)isolating nucleic acid molecules from the isolated population of memoryB cells; (d) amplifying nucleic acids encoding the variable heavy chainsand the variable light chains from the nucleic acids; (e) expressing thevariable heavy chains and the variable light chains from the nucleicacids to produce antibody molecules from the variable heavy chains andthe variable light chains; and (f) selecting an monoclonal antibody thatspecifically binds to the target antigen.

Some embodiments, include a method for isolating the repertoire of Bcells specific for a target antigen from a subject, including: (a)isolating a population of memory B cells from a subject that has beenexposed to the target antigen, wherein the memory B cells areCD19+IgA-IgD-IgM- B cells; (b) contacting the isolated population ofmemory B cells with an effective amount of IL-21, IL-2 and CD40; and (c)selecting B cells from the population that express antibodies thatspecifically bind the target antigen, thereby isolating the repertoireof B cells specific for the target antigen from the subject.

In several embodiments, the target antigen is an antigen from apathogen, such as a virus, a fungus, a parasite or a bacteria. Theantigen can be from a virus, for example, human immunodeficiency virus(HIV), such as HIV-1. In some embodiments, the antigen can be HIV-1gp41. In some embodiments wherein the target antigen is a HIV antigen,the subject is as subject infected with a HIV, such as HIV-1. In otherembodiments, the target antigen is a cancer antigen. In some suchembodiments, the subject is a subject with cancer. In still otherembodiments, the target antigen is a self-antigen. For example, in someembodiments, the target antigen is a toxin, such as a bacterial toxin,for example, anthrax toxin. In additional embodiments, the targetantigen is an antigen included in a vaccine. The person of ordinaryskill in the art will appreciate that the target antigen can be anyantigen to which a subject is capable of producing a B cell responsethat results in the production of memory B cells that produce antibodythat specifically binds to the target antigen.

In some embodiments, the isolated population of memory B cells isrepresentative of the repertoire of B cells in the subject that arespecific to the target antigen.

In some embodiments of the disclosed methods, contacting the isolatedpopulation of memory B cells with CD40L includes incubating the isolatedmemory B cells with a feeder cell that expresses the CD40L.

In additional embodiments of the disclosed methods, contacting theisolated population of isolated memory B cells with CD40L, IL-2 andIL-21 includes incubating the isolated population of memory B cells withCD40L, IL-2 and IL-21 for about 10 to about 15 days, such as about 13days.

EXAMPLES

The following examples are provided to illustrate particular features ofcertain embodiments, but the scope of the claims should not be limitedto those features exemplified.

Example 1 Isolation and Characterization of Broadly NeutralizingMPER-Specific Monoclonal Antibodies

This example illustrates isolation and characterization of HIV-141-specific binding antibodies from a subject infected with HIV-1.

Abstract. Characterization of human monoclonal antibodies has providedconsiderable insight into mechanisms of broad HIV-1 neutralization. Thisexample described isolation of an HIV-1 gp41 membrane-proximal externalregion (MPER)-specific antibody, named 10E8, which neutralizes ˜98% oftested viruses. An analysis of sera from 78 healthy HIV-1-infecteddonors demonstrated that 27% contained MPER-specific antibodies and 8%contained 10E8-like specificities. In contrast to other neutralizingMPER antibodies, 10E8 did not bind phospholipids, was not autoreactive,and bound cell-surface envelope. The structure of 10E8 in complex withthe complete MPER revealed a site-of-vulnerability including a narrowstretch of highly conserved gp41-hydrophobic residues and a criticalArg/Lys just prior to the transmembrane region. Analysis of resistantHIV-1 variants confirmed the importance of these residues forneutralization. The highly conserved MPER is a target of potent,non-self-reactive neutralizing antibodies, suggesting that HIV-1vaccines should aim to induce antibodies to this region of HIV-1 Env.

Introduction. Induction of an antibody response capable of neutralizingdiverse HIV-1 isolates is a critical goal for vaccines that protectagainst HIV-1 infection. Potentially the greatest obstacle to achievingthis goal is the extraordinary diversity that develops in the target ofneutralizing antibodies, the envelope glycoprotein (Env). Althoughvaccines have thus far failed to induce broadly neutralizing antibodyresponses, there are examples of chronically infected patients with serathat neutralize highly diverse HIV-1 isolates. These individuals provideevidence that it is possible for the human antibody response toneutralize highly diverse strains of HIV-1, though the mechanisms bywhich such responses are induced or mediated remain incompletelyunderstood (Haynes et al., Nat Biotechnol 30, 423-433, 2012; Walker etal., Curr Opin Immunol 22, 358-366, 2010).

Recently, isolation and characterization of human monoclonal antibodiesfrom cells of chronically infected patients have provided considerableadvances in understanding the specificities and mechanisms underlyingbroadly neutralizing antibody responses to HIV-1. Env exists on thevirion and infected-cell surface as a trimer of heterodimers made up ofgp120 and gp41 subunits. For some time, only a small number of broadlyneutralizing monoclonal antibodies (mAbs) had been isolated consistingof one antibody that binds the CD4-binding site on gp120 (b12), one thatbinds a glycan configuration on the outer domain of gp120 (2G12) andthree that bind the membrane-proximal external region (MPER) on gp41(2F5, Z13e1, and 4E10; Zwick et al., J Virol 75, 10892-10905, 2001;Burton et al., Science 266, 1024-1027, 1994; Muster et al., J Virol 67,6642-6647, 1993). More recently, considerably more broad and potentantibodies have been discovered that target the CD4-binding site of theenvelope protein (for which VRC01 is a prototype; Bonsignori et al., JVirol 86, 4688-4692, 2012; Wu et al, Science 333, 1593-1602, 2011;Scheid et al., Science 333, 1633-1637, 2011; Wu et al., Science 329,856-861, 2010) and glycan containing regions of the V1/V2 and V3 regionsof gp120 (for which PG9 and PGT128 are prototypes; Walker et al., PLoSPathog 6, e1001028, 2010; Walker et al., Nature 477, 466-470, 2011;Bonsignori et al., J Virol 85, 9998-10009, 2011; Walker et al., Science326, 285-289, 2009). The specificities of these new antibodies areproviding important information regarding antigen targets on Env towhich the humoral immune response might be directed to mediate broad andpotent neutralization. However, evidence for these specificities in manychronically infected patients within the cohort is lacking, suggestingthat broad and potent neutralization may be mediated by otherspecificities.

This example describes isolation of a broad and potent gp41MPER-specifichuman mAb, 10E8, from an HIV-1-infected individual with highneutralization titers. 10E8 is among the most broad and potentantibodies thus far described, and lacks many of the characteristicspreviously thought to limit the usefulness of MPER-specific antibodiesin vaccines or passive therapies, including lipid binding andautoreactivity. In addition, the crystal structure of 10E8, along withbiochemical binding studies, demonstrate that the breadth of 10E8 ismediated by its unique mode of recognition of a structurally conservedsite-of-vulnerability within the gp41MPER.

10E8 isolation and neutralizing properties. To understand thespecificities and binding characteristics that underlie a broadlyneutralizing antibody response techniques were developed that permittedisolation of human monoclonal antibodies without prior knowledge ofspecificity. Serum from one donor, N152, exhibited neutralizing breadthand potency in the top 1% of the cohort against a 20 cross-cladepseudovirus panel (FIG. 17; Doria-Rose et al., J Virol 84, 1631-1636,2010). Peripheral blood CD19+IgM-IgD-IgA- memory B cells from thispatient were sorted and expanded for 13 days with IL-2, IL-21, andCD40-ligand expressing feeder cells. The supernatants of ˜16,500 B cellcultures were screened and IgG genes from wells with neutralizationactivity were cloned and re-expressed (Tiller et al., Journal ofimmunological methods 329, 112-124, 2008) and two novel antibodies (10E8and 7H6) were isolated.

Nucleotide sequence analysis of DNA encoding 10E8 and 7H6 revealed thatboth were IgG3 antibodies and were somatic variants of the same IgGclone. These antibodies were derived from IGHV3-15*05 and IGLV3-19*01germline genes, and were highly somatically mutated in variable genes ofboth heavy chain (21%) and lambda light chain (14%) compared togermline. These antibodies also possessed a long heavy-chaincomplementarity-determining region (CDR H3) loop composed of 22 aminoacids (FIG. 1A). The heavy chains of 10E8 and 7H6 were identical andthere were only two residue differences in the light chain (FIG. 6).

To assess neutralization activity of the clonal variants, they wereinitially tested against 5 Env-pseudoviruses (FIG. 17A), and mAb 10E8was selected for further study. To determine if the neutralizationactivity of 10E8 was representative of the overall neutralizationspecificity present in patient N152 donor serum, the neutralizationpanel was expanded to 20 Env-pseudoviruses, and 10E8 was tested inparallel with N152 donor serum. Although there were some similarities inthe pattern of neutralization of highly resistant variants, acorrelation of the neutralization IC50 of mAb 10E8 and ID50 of N152serum did not achieve statistical significance (p=0.11; FIGS. 7 and17B). This finding suggests that although 10E8 may play a major role,the full breadth of neutralization of by N152 serum is likely mediatedby an amalgam of 10E8-like or other antibodies.

To compare the neutralization potency and breadth of 10E8 with otherbroadly neutralizing anti-HIV-1 antibodies, 10E8 was then tested in a181-isolate Env-pseudovirus panel in parallel with 4E10, 2F5, VRC01,NIH45-46, 3BNC117, PG9, and PG16 (FIG. 1B and FIGS. 17C-17F). At an IC50below 50 μg/ml, 10E8 neutralized 98% of the tested pseudovirusescompared to 98% for 4E10 and 89% for VRC01. However, at an IC50 below 1μg/ml, 10E8 neutralized 72% of the tested viruses compared to 37% for4E10. The median and geometric mean IC50 values for 10E8 were below 1ug/ml. Thus, 10E8 mediates broad and potent neutralization against alarge range of viruses and the potency is comparable to some of the bestavailable monoclonal antibodies.

10E8 epitope specificity and binding. To map the epitope of the 10E8antibody, binding to different subregions of Env by enzyme-linkedimmunosorbent assay was tested (ELISA). 10E8 bound strongly to gp140,gp41, and the 4E10-specific MPER peptide, but not to gp120 (FIG. 2A). Tofurther map the 10E8 epitope within the MPER, binding of 10E8 tooverlapping peptides corresponding to the 2F5 (656-671), Z13e1(666-677), and 4E10 (671-683) specificities was examined. 10E8 bound tothe full MPER and the 4E10-specific peptides, but not 2F5- orZ13e1-specific peptides. Within the 4E10 epitope, when a peptide with atruncated C-terminus was tested, 4E10.19 (671-680), 10E8 binding wasweakened considerably, suggesting that the three terminal amino acids ofthe MPER, Tyr681, 11e682, and Arg683, were crucial for 10E8 binding(FIG. 8A). Consistent with these results, only the full MPER and4E10-specific peptides blocked 10E8-mediated neutralization of thechimeric C1 virus, which contains the HIV-2 Env with the HIV-1 MPER(FIG. 8B). Taken together, these data suggest that the minimal 10E8epitope is located within residues 671-683 of the MPER althoughadditional contacts toward the amino terminus of the MPER could not beexcluded.

To more precisely map the epitope of 10E8, a panel of alanine mutantpeptides scanning MPER residues 671-683 was used to block 10E8neutralization of the chimeric C1 virus in a TZM-b1 assay (FIG. 2B;Brunel et al., J Virol 80, 1680-1687, 2006). For these assays, the basepeptide was 4E10.22 MPER peptide (CNWFDITNWLWYIRKKK; SEQ ID NO: 14) withthe indicated alanine substitutions. MPER peptides with alaninesubstitutions at Trp672, Phe673 or Thr676 failed to block 4E10 or 10E8neutralization, suggesting that these residues are critical for both4E10 and 10E8 binding. Residue Asn671 and residue Arg683, both of whichare not required for 4E10 binding, were found to be critical for 10E8binding and neutralization (FIGS. 2B and 18). The ability of 10E8 toneutralize HIV-1_(JR2) pseudoviruses with alanine substitutions in MPERresidues 660-683 was also tested (FIG. 19). Consistent with the effectsof alanine substitutions on peptide binding, residues Asn671 and Arg683were critical for 10E8, but not 4E10, neutralization. Individual alaninesubstitutions at residues 671-673, 680 and 683 resulted in reducedneutralization sensitivity to 10E8 most apparent at the IC₉₀ levelrather than the IC50 level. Although the mechanism for this phenomenonis unclear, a similar effect has been observed previously when MPERmutations cause partial resistance to 4E10 (Zwick et al., J Virol 79,1252-1261, 2005). Taken together, these results suggested that 10E8recognized a novel epitope, which overlaps the known 4E10 and Z13e1epitopes, but differs in a critical dependence on binding to Asn671 andArg683, the last residue of the MPER.

Whether the greater neutralization potency of 10E8 compared to otherMPER antibodies was a result of higher binding affinity to the MPER wasnext investigated. Capture of a biotinylated peptide encompassing thefull MPER (656-683) to a surface-plasmon resonance chip allowed thebinding kinetics of Fabs 10E8, 2F5 and 4E10 to be examined. In contrastto its higher neutralization potency, the K_(D) of 10E8 to this MPERpeptide was weaker than that of 2F5 and 4E10; 17 nM for 10E8 versus 3.8nM for 2F5 and 0.74 nM for 4E10 (FIG. 9). Therefore, the affinity of10E8 for the MPER in a soluble peptide format did not explain itsgreater neutralization potency compared to other MPER-specificantibodies.

Prevalence of 10E8-like antibodies. The prevalence of MPER-specific and10E8-like neutralizing antibodies in the cohort of HIV-1-infected donorswas next investigated. Next, 78 sera from the cohort with aneutralization ID50>100 against at least one pseudovirus in a 5-virusmini-panel were selected (Doria-Rose et al., J Virol 84, 1631-1636,2010). The median time since diagnosis of these donors was 13.5 years,median CD4 count was 557 cells/μl, median plasma HIV RNA 5573 copies/ml,and they were not receiving antiretrovirals. Neutralization against theHIV-2/HIV-1 chimera C1 was tested (FIG. 20). Of 78 sera, 21 exhibitedneutralization activity against the HIV-2/HIV-1 C1 virus (FIG. 21). Tomap the region that was targeted by these sera, neutralization wasmeasured using 7 HIV-2/HIV-1 chimeras containing subdomains of the MPER(FIG. 20; Gray et al., J Virol 81, 6187-6196, 2007). Of the 21 sera withneutralization activity against the entire MPER, 8 exhibited aneutralization pattern similar to that observed for 10E8, which entailedneutralization of only those HIV-2/HIV-1 chimeric viruses that containedthe terminal residue of the MPER Arg683 (C4, C4GW and C8; FIG. 21). Tofurther confirm these results, peptides corresponding to differentportions of the MPER to block sera neutralization of the HIV-2/HIV-1chimera C1 were used (FIG. 22). Of the 8 sera found to have a 10E8-likepattern based upon neutralization of the chimeras, 3 were blocked bypeptides consistent with 10E8-like activity. An additional 3 of the 810E8-like sera were blocked by peptides in a pattern consistent with acombination of 10E8 and Z13e1-like antibodies. The 6 patients whose serahad 10E8-like activity did not differ from the remaining 72 patientswith regard to clinical course or HIV neutralization (legend; FIG. 10,legend). Overall, 27% of the tested patient sera exhibited anti-MPERneutralizing activity. This prevalence is considerably higher thanobserved in prior work, possibly related to selection of donors withknown neutralizing activity (Gray et al., J Virol 83, 8925-8937, 2009;Tomaras et al., J Virol 85, 11502-11519, 2011; Morris et al., PLoS ONE6, e23532, 2011; Gray et al., J Virol 83, 11265-11274, 2009). Further,8% of the tested sera had 10E8-like antibodies (FIG. 10), suggestingthat 10E8-like antibodies are not rare.

Analysis of 10E8 autoreactivity. A property common to the previouslycharacterized MPER mAbs 2F5 and 4E10 is that they cross-react withself-antigens (Haynes et al., Science 308, 1906-1908, 2005). Inaddition, binding to both the cell membrane and the Env trimer isthought to be important for optimal neutralization by these antibodiesand this autoreactivity may be an obstacle to the elicitation of similarantibodies by a vaccine (Haynes et al., Science 308, 1906-1908, 2005;Alam et al., Proceedings of the National Academy of Sciences of theUnited States of America 106, 20234-20239, 2009). Surface plasmonresonance analysis showed that 10E8 did not bind to anionicphospholipids, such as phosphatidyl choline-cardiolipin (PC-CLP) andphosphatidyl choline-phosphatidyl serine (PC-PS) liposomes (FIG. 3A).10E8 also did not bind HEp-2 epithelial cells, in contrast to 2F5 and4E10 that bound in a cytoplasmic and nuclear pattern (FIG. 3B).Additionally, 10E8 did not bind autoantigens, such as Sjogren's syndromeantigens A and B, Smith antigen, ribonucleoprotein, scleroderma 70antigen, Jo1 antigen, centromere B and histone (FIG. 23). Takentogether, these results suggest that 10E8, in contrast to other MPERantibodies, is not autoreactive.

Virion accessibility of 10E8. The 2F5 and 4E10 antibodies have beenshown to bind relatively poorly to the HIV-1 envelope spike on thesurface of infected cells or to cell-free virions, and react moreefficiently after Env engagement of the CD4 receptor (Chakrabarti etal., J Virol 85, 8217-8226, 2011). Binding to cleaved, full-lengthenvelope spikes on HIV_(JRFL) transfected cells was measured (FIG. 11A).Although 10E8 bound less efficiently than other antibodies such as VRC01or F105, where accessibility is not an issue, it bound more efficientlythan either 2F5 or 4E10. In contrast to results of alanine substitution,a mutation in the 4E10 (F673S) region in full-length HIV envelope spikesenhanced 10E8 binding although the mechanism remains unclear. A mutationin the 2F5 (K665E) region had no influence on 10E8 binding. These datasuggest that 10E8 has modestly greater access to the MPER epitope on thecell surface than either 2F5 or 4E10.

To assess binding to cell-free virus, virions were incubated withantibody, washed out unbound antibody, and tested neutralization(Chakrabarti et al., J Virol 85, 8217-8226, 2011; Frey et al.,Proceedings of the National Academy of Sciences of the United States ofAmerica 105, 3739-3744, 2008; Rathinakumar et al., J Virol 86,1820-1831, 2012). During washing, antibodies that cannot access theirEnv target on free virions will be largely removed and thereforeneutralization will be diminished. As a control, neutralization of theHXBc2 isolate was not diminished by washing, because the MPER region isaccessible on this laboratory adapted isolate (Chakrabarti et al., JVirol 85, 8217-8226, 2011). Washing also had little impact onneutralization of JRFL by VRC01. Consistent with prior work, 2F5 and4E10 neutralization of most virus isolates tested was substantiallydiminished after washing (FIG. 11B). In contrast to 2F5 and 4E10,washing had a smaller effect on 10E8 neutralization of most virusestested, as measured by the area under the curve or analysis of thefold-change in neutralization at a fixed inhibitory concentration (FIG.11C). Although 10E8 is not fully able to access its epitope on thenative viral spike similarly to VRC01, under most experimentalconditions tested it was better able to access its epitope than either2F5 or 4E10.

Structure of 10E8-gp41 complex. To provide an atomic-level understandingof the interaction of 10E8 with HIV-1, the antigen-binding fragment(Fab) of 10E8 in complex with a peptide encompassing the entire28-residue gp41MPER (residues 656-683) was crystallized. Monocliniccrystals diffracted to 2.1 Å resolution, and structure solution andrefinement to R_(cryst)=18.01% (R_(free)=21.76%) revealed two complexesin the asymmetric unit (heretofore referred to as complexes 1 and 2)(FIG. 24). Overall, 10E8 bound to one face of the MPER peptide, whichformed two helices, each 15-20 Å in length and oriented 100° relative toeach other (FIG. 4A). Electron density was observed for the entire MPER,ranging from Asn656-Arg683 (Leu660-Arg683 for complex 2), with thehighest degree of ordered density observed from residue Trp666 withinthe N-terminal helix through to Arg683 of the C-terminal helix (FIG.12). Analysis of main-chain dihedral angles (FIG. 25) indicated that theN-terminal α-helix extends from residue Asn657 to Ala667, tightens intoa 3₁₀-helix between residues Ser668 and Leu669, before turning atresidues Trp670 and Asn671. The C-terminal α-helix, capped by Asn671,starts at residue Trp672 and extends to residue Arg683, the finalresidue of the MPER (FIG. 4A,B).

The 10E8 antibody contacts the gp41MPER primarily through its heavychain, although crucial contacts are also mediated by the light chainCDR L3 (FIGS. 4C and 26-28). Three predominant loci of interaction areobserved between the antibody and gp41 (FIGS. 29-30): One betweenresidues of the tip of the CDR H3 loop and the tip of the C-terminalhelix of the peptide, a second between residues of the CDR H2 loop andresidues of the hinge region of the peptide, and a third at the junctureof the three heavy chain CDR loops and the light chain CDR L3, whichform a hydrophobic cleft that holds residues of the beginning of theMPER C-terminal helix (FIG. 4B).

10E8-gp41 interface. To complement the results observed for themutagenesis of the highly conserved 10E8 epitope (FIGS. 4D and 18), eachresidue of the 10E8 paratope, as determined from the crystal structure,was individually mutated to alanine and the resulting 25 10E8 variantsassessed for affinity to a soluble MPER peptide. Overall, the mostpronounced effects of the alanine mutations on the binding affinity of10E8 to a soluble MPER peptide occurred within residues of the CDR H3loop, though mutations within the hydrophobic cleft also showedsubstantial effect (FIGS. 4E, 13 and 31). 10E8 residues identified byalanine scan as critical for the interaction with gp41 stretched fromthe cleft all the way to the tip of the CDR H3 (FIG. 4E) and weremirrored by a corresponding stretch of gp41 residues which substantiallyaffected 10E8 binding when mutated to alanine (FIG. 4F).

The same panel of 10E8 alanine mutations was tested for neutralizationpotency against a panel of five Env-pseudoviruses that included bothTier 1 and Tier 2 viruses (FIG. 32). Similar to the binding data,residues of the 10E8 CDR H3 had dramatic effects on neutralization, asdid residues of the hydropohobic cleft (FIG. 4g ). Generally, K_(D)s ofparatope mutants correlated with neutralization (FIG. 14). Backboneinteractions (on both 10E8 and gp41) also contribute to the interface,especially between the CDR H2 of 10E8 and the hinge region of the MPER,though these are silent in alanine scan analyses. Overall, 10E8 utilizesa narrow band of residues (˜20×5 Å) that stretches from the CDR H1 andH2 and extends along most of the CDR H3 to recognize a string of highlyconserved hydrophobic gp41 residues, and a critical charged residue,Arg/Lys683, that occurs just prior to the transmembrane region (FIG.4F,H).

A Conserved gp41-neutralization determinant. Several structures ofneutralizing antibodies in complex with the MPER of gp41 have beenreported previously, including those for antibodies 2F5, Z13e1 and 4E10(FIG. 15A; Julien et al., J Mol Biol 384, 377-392, 2008; Cardoso et al.,J Mol Biol 365, 1533-1544, 2007; Cardoso et al., Immunity 22, 163-173,2005; Ofek et al., J Virol 78, 10724-10737, 2004; Pejchal et al., JVirol 83, 8451-8462, 2009). The MPER adopts divergent loop conformationswhen bound by 2F5 and Z13e1 and an α-helix when bound by 4E10.Comparison of 2F5, Z13e1, and 4E10 epitopes with 10E8-bound gp41revealed that only the 4E10 epitope has similar secondary structure,with superposition yielding an RMSD of 2.49 Å for all atoms of residues671-683 and 0.98 Å for main-chain atoms (FIGS. 15B and 33).

To compare further the recognition of 10E8 and 4E10, their angles ofepitope approach were examined. As shown in FIGS. 15C-15F, alignment ofthe recognized MPER helix places 10E8 and 4E10 into similar overallspatial positions. The relative orientations of the recognized helix andthe heavy and light chains of the two antibodies, however, differdramatically. With 10E8, the C-terminal helix is perpendicular to theplane bisecting heavy and light chains (FIGS. 15C,E); with 4E10, therecognized helix is at the interface between heavy and light chains(FIG. 15D,F). Perhaps relevant to this, 10E8 utilizes CDR loops almostexclusively in its recognition of gp41, while 4E10 incorporatessubstantial β-strand interactions with gp41 at the interface between theheavy and light chains.

The differing modes of 10E8 and 4E10 recognition of the conservedC-terminal MPER helix result in a substantial difference in theproportion of the recognized helical face: 10E8 contacts roughly a thirdof the helical face, while 4E10 contacts over half (FIGS. 15G, 15H and34-35). The smaller contact surface of 10E8 may provide an explanationfor the reduced recognition of lipid surfaces by 10E8 versus4E10—providing a potential structure-based explanation for reducedautoreactivity of 10E8.

Sequence variation and 10E8 neutralization. To place the specificity andstructural data into the context of known variation of the MPER, viralsequences with resistance to neutralization by 10E8 were analyzed (FIG.5A). Of the 183 viruses tested, only three were highly resistant to 10E8with IC50 >50 μg/ml. Each of these viruses had substitutions atpositions found to affect neutralization by alanine scanning (Asn671,Trp672, Phe673, and Trp680). Plasma virus of the patient N152, from whom10E8 was cloned, is also likely resistant to 10E8 mediatedneutralization (Wu et al., J Virol 86, 5844-5856, 2012). Sequenceanalysis of plasma viral RNA revealed rare substitutions at positionsTrp680 and Lys/Arg683 (FIG. 5A). These residues are typically highlyconserved with variation only occurring in 1.17% of 3,730 HIV Envsequences in the Los Alamos Database (hiv.lanl.gov). When thesubstitutions for the 3 resistant viruses and the patient viruses wereplaced on the background of the sensitive JR2 virus, substitutions atAsn671Thr, Trp672Leu, and Phe673Leu had a modest effect on the IC50 butraised the IC80 above 20 μg/ml. In the structural analysis, directcontacts with 10E8 were not observed at position 671 suggesting theeffects on neutralization of Thr or Ala substitutions at this positionare mediated by conformational or other effects within gp41. Thecombination of Trp672Leu and Phe673Leu conferred high-level resistanceat the IC50 and IC80 level. Changes corresponding to the patient'sdominant circulating virus had a similar effect. Although Lys/Arg683Glnalone conferred resistance at the IC80 level, together Trp680Arg andLys/Arg683Gln resulted in greater resistance to 10E8 (FIG. 5A). Whentaken together with the analysis of the 10E8 paratope, these datasuggest that in addition to Trp672, Phe673, and Trp680 found in the 4E10epitope, the additional 10E8-bound residue Lys/Arg683 is critical toneutralization. In addition to other differences in binding based uponstructural analyses noted above, it is possible that the additionalpotency of 10E8 compared to 4E10 against naturally occurring viralvariants may be mediated through binding of highly conserved residuesTrp680 and Lys/Arg683 that directly interact with the 10E8 CDRH3.

Discussion. 10E8 is a broad and potent neutralizing antibody withimportant implications for efforts to stimulate such antibodies withvaccines. Previous MPER antibodies were somewhat limited in potency, andhad a more limited ability to access MPER on Env of primary isolates. Inaddition, lipid binding and autoreactivity were thought to becharacteristics of MPER antibodies and important obstacles to theirelicitation by vaccines. However, 10E8 lacks each of thesecharacteristics. In addition, antibodies with a similar specificity werenot rare in the chronically infected cohort. This suggests that10E8-like antibodies were not deleted from the repertoire because ofautoreactivity. These results further suggest that 10E8-like antibodiesmight be raised in a larger fraction of HIV-uninfected persons receivinga vaccine designed to elicit these antibodies without the B cell defectsof chronic HIV infection. Design of such a vaccine will likely requirenot only presentation of an intact 10E8 epitope but also use of aplatform sufficiently immunogenic to drive the evolution of 10E8-likeantibodies.

The extraordinary breadth and potency of 10E8 appears to be mediated byits ability to bind highly conserved residues within MPER. Although theepitope of 10E8 overlaps those of known mAbs such as 4E10, it differs inrecognition surface, angle of approach, lipid binding, andself-reactivity. Alanine scanning, structural analysis, and paratopeanalysis each indicate that 10E8 makes crucial contacts with highlyconserved residues Trp672, Phe673, Trp676 and Lys/Arg683. Theextraordinary breadth of some potent mAbs, for example that bind the CD4binding site, is thought to be conferred by blocking a functionallyimportant site that is critical for viral entry. Whether 10E8 impairsEnv function or simply acts by binding highly conserved residues remainsto be determined. Nonetheless, the breadth and potency of 10E8demonstrates a conserved site of gp41 vulnerability (FIG. 5B) that is animportant target antigen for HIV neutralization and that will likelyreinvigorate interest in MPER-based HIV vaccine design.

Methods

Methods summary. Peripheral blood CD19+IgM-IgD-IgA- B cells were sortedby flow cytometry, plated at 4 cells per well, and expanded withcytokines and feeder cells. B-cell culture supernatants were screened bymicroneutralization against HIV_(MN.03) and HIV_(Bal.26) pseudoviruses.IgG genes from wells with neutralization activity were cloned andre-expressed in 293T cells. Breadth of neutralizing activity wasconfirmed against a 181-isolate Env-pseudovirus panel. Specificity wasdetermined by alanine scanning peptides and mutant-Env pseudoviruses.Lipid binding and autoreactivity of 10E8 were measured by surfaceplasmon resonance, indirect immunofluorescence on HEp-2 cells and beadarrays. Binding of HIV envelopes on transfected 293 cells was detectedby flow cytometry. Following pre-incubation with antibody, the impact ofwashing virions prior to infecting TZM-b1 cells was used to measureaccess to viral MPER. The frequency of HIV-1⁺ sera with a givenspecificity was measured by the ability to neutralize HIV-2/HIV-1chimeras containing portions of the MPER. Successful co-crystallizationof 10E8 with gp41 was obtained when a peptide encompassing the entire28-residue gp41MPER (residues 656-683). Structure determination revealedtwo complexes in the crystal asymmetric unit. Analysis of differencesbetween the two complexes enabled essential interactions to bediscerned. The paratope, as defined by residues in the antibody thatshowed reduced solvent accessibility when complexed by gp41, wassubjected to comprehensive alanine scan, with each of the 25 10E8alanine mutants assessed by SPR for recognition of gp41 and byneutralization on a panel of 5 pseudotyped viruses. The sequence of thepatient plasma viral RNA was derived using limiting dilution RT-PCR.

Study patients. Plasma and peripheral blood mononuclear cells (PBMC)were selected from the HIV-1-infected patients enrolled in the NationalInstitute of Health under a clinical protocol approved by theInvestigational Review Board in the National Institute of Allergy andInfectious Diseases (NIAID-IRB). All participants signed informedconsent approved by the NIAID-IRB. The criteria for enrollment were asfollows: having a detectable viral load, a stable CD4 T-cell count above400 cells/μl, being diagnosed with HIV infection for at least 4 years,and off ARV treatment for at least 5 years. Based on the locations ofcurrent and former residences, all patients were presumed to be infectedwith Glade B virus. Donor N152 was selected for B cell sorting andantibody generation because his serum neutralizing activity is among themost potent and broad in the cohort. He is a slow progressor based oncriteria described previously (Migueles et al., Immunity 29, 1009-1021,2008). At the time of leukapheresis, he had been infected with HIV-1 for20 years, with CD4 T-cell counts of 325 cells/μl, plasma HIV-1 RNAvalues of 3,811 copies/ml and was not on antiretroviral treatment.

Memory B-cell staining, sorting and antibody cloning. Staining andsingle-cell sorting of memory B cells were performed as follows. PBMCsfrom HIV-1 infected donor N152 were stained with antibody cocktailconsisting of anti-CD19-PE-Cy7 (BD Bioscience), IgA-APC (JacksonImmunoResearch Laboratories Inc.), IgD-FITC (BD Pharmingen), and IgM-PE(Jackson ImmunoResearch Laboratories Inc.) at 4° C. in dark for 30 min.The cells were then washed with 10 ml PBS-BSA buffer and resuspended in500 μl PBS-BSA. 66,000 CD19+IgA-IgD-IgM- memory B cells were sortedusing a FACSAria III cell sorter (Becton Dickinson) and resuspended inIMDM medium with 10% FBS containing 100 U/ml IL-2, 50 ng/ml IL-21 and1×10⁵/ml irradiated 3T3-msCD40L feeder cells (Kershaw et al., Cancer Res61, 7920-7924, 2001). B cells were seeded into 384-well microtiterplates at a density of 4 cells/well in a final volume of 50 μl. After 13days of incubation, 40 μl of culture supernatants from each well werecollected and screened for neutralization activity using a highthroughput micro-neutralization assay against HIV-1_(MN.03) andHIV-1_(Bal.26). B cells in each well were lysed with 20 μl lysis buffercontaining 0.25 μl of RNase inhibitor (New England Biolabs Inc.), 0.3 μlof 1M Tris pH8 (Quality Biological Inc.) and 19.45 μl DEPC-treated H₂O.The plates with B cells were stored at −80° C.

The variable region of the heavy chain and the light chain of theimmunoglobulin gene were amplified by RT-PCR from the wells that scoredpositive in both the HIV-1_(MN.03) and HIV-1_(Bal.26) neutralizationassay. The cDNA product was used as template in the PCR reaction. Inorder to amplify the highly somatically mutated immunoglobulin gene, twosets of primers as described previously (Tiller et al., Journal ofimmunological methods 329, 112-124, 2008) were used in two independentPCRs. One set of primers consisted of the forward primers and thereverse primers specific for the leader region and constant region ofIgH, Igκ or Igλ, respectively. The other set of primers consisted of theforward primer mixes specific for FWR1 and respective reverse primersspecific for the IgH, Igκ and Igλ J genes. All PCRs were performed in96-well PCR plates in a total volume of 50 μl containing 20 nM eachprimer or primer mix, 10 nM each dNTP (Invitrogen), 10 μl 5× Q-solution(Qiagen) and 1.2 U HotStar Taq DNA polymerase (Qiagen). From thepositive PCR reactions, pools of the VH or VL-region DNA were ligated toa pCR2.1-Topo-TA vector (Invitrogen) for sequencing before cloning intothe corresponding Igγ1, Igκ and Igλ expression vector. 10 μg of heavyand light chain plasmids, cloned from the same well and combined in allpossible heavy and light chain pairs, were mixed with 40 μl FuGENE 6(Roche) in 1500μl DMEM (Gibco) and co-transfected into 293T cells. Thefull-length IgG was purified using a recombinant protein-A column (GEHealthcare).

Neutralization assays. Neutralization of the monoclonal antibodies wasmeasured using single-round HIV-1 Env-pseudoviruses infection of TZM-b1cells (Li et al., J Virol 79, 10108-10125, 2005). HIV-1Env-pseudoviruses were generated by co-transfection of 293T cells withpSG3ΔEnv backbone containing a luciferase reporter gene and a secondplasmid that expressed HIV-1 Env. At 72 hours post-transfection,supernatants containing pseudovirus were harvested and frozen at −80° C.until further use. In the neutralization assay, 10 μl of 5-fold seriallydiluted patient serum or mAb was incubated with 40 μl pseudovirus in a96-well plate at 37° C. for 30 minutes before addition of TZM-b1 cells.After 2 days of incubation, cells were lysed and the viral infectivitywas quantified by measuring luciferase activity with a Victor Lightluminometer (Perkin Elmer). The 50% inhibitory concentration (IC50) wascalculated as the antibody concentration that reduced infection by 50%.Antibody epitopes were mapped using HIV-1 JR2 MPER alanine mutantpseudoviruses in a TZM-b1 assay.

HIV-2/HIV-1 chimera neutralization. HIV-2/HIV-1 C1 chimera (HIV-2 virus7312A with HIV-1 gp41MPER; Gray et al., J Virol 81, 6187-6196, 2007) wasused in the competition assay. A fixed concentration of MPER peptide wasincubated with serially diluted 2F5, 4E10, Z13e1 or 10E8 antibody at 37°C. for 30 minutes before incubation with HIV-2/HIV-1 C1 chimera.Wild-type HIV-2 virus 7312A was used as a control. Antibody epitopemapping was completed by adding 10 μl 10E8 mAb to 5 μl serial dilutionsof 4E10 peptide or its alanine mutants at 37° C. for 30 minutes prior tothe addition of HIV-2/HIV-1 C1 chimera. The degree to which peptidesblocked antibody-mediated neutralization was calculated as the foldchange in the IC50 value of the antibody in the presence of 4E10 alaninemutants compared to the wild-type peptide. The precise binding regionwithin the MPER targeted by patient serum or antibodies was determinedusing the HIV-2/HIV-1 chimeras containing different portions of HIV-1MPER, such as C1 (HIV-2 Env with HIV-1 MPER), C1C (HIV-2 Env with GladeC MPER), C3 (HIV-2 Env with 2F5 epitope), C4 (HIV-2 Env with 4E10epitope), C6 (HIV-2 Env with short 4E10 epitope NWFDIT), C7 (HIV-2 Envwith short 2F5 epitope ALDKWA) and C8 (HIV-2 Env with both Z13 and 4E10epitope). 5-fold diluted patient serum or mAb was incubated with chimerain a 96-well plate at 37° C. for 30 minutes before addition of TZM-b1cells. The specificities within patient sera were confirmed by blockingneutralization of the C1 chimera with 25 μg/ml of 2F5, 4E10, MPER,Bal.V3, control peptide, or 50 μg/ml of Z13 peptide.

ELISA assays. Each antigen at 2 μg/ml was coated on 96-well platesovernight at 4° C. Plates were blocked with BLOTTO buffer (PBS, 1% FBS,5% non-fat milk) for one hour at room temperature (RT), followed byincubation with antibody serially diluted in disruption buffer (PBS, 5%FBS, 2% BSA, 1% Tween-20) for one hour at room temperature. 1:10,000dilution of horseradish peroxidase (HRP)-conjugated goat anti-human IgGantibody was added for one hour at room temperature. Plates were washedbetween each step with 0.2% Tween 20 in PBS. Plates were developed using3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma) and read at 450 nm.

Autoreactivity assays. Binding of 10E8 to phospholipid was measured bySPR conducted on a BIACORE® 3000 instrument and data analyses wereperformed using the BIAevaluation® 4.1 software (BIACORE®) as describedpreviously (Alam et al., Proc. Natl. Acad. Sci. U.S.A., 106,20234-20239, 2009). Phospholipid-containing liposomes were captured on aBIACORE® L1 sensor chip, which uses an alkyl linker for anchoringlipids. Before capturing lipids, the surface of the L1 chip was cleanedwith a 60-s injection of 40 mM octyl-β-D-glucopyranoside, at 100μl/minute, and the chip and fluidics were washed with excess buffer toremove any traces of detergent. mAbs were then injected at 100 μg/ml ata flow rate of 30 μl/min. After each Ab injection, the surface was againcleaned with octyl β-D-glucopyranoside, and 5-s injections of each 5 mMHCl, then 5 mM NaOH, to clean any adherent protein from the chip.

Reactivity to HIV-1 negative human epithelial (HEp-2) cells wasdetermined by indirect immunofluorescence on slides using Evans Blue asa counterstain and FITC-conjugated goat anti-human IgG (Zeus Scientific,Raritan N.J.; Haynes et al., Science 308, 1906-1908, 2005). Slides werephotographed on a Nikon Optiphot fluorescence microscope. Regarding FIG.3B, kodachrome slides were taken of each MAb binding to HEp-2 cells at a32 second exposure, and the slides scanned into digital format. TheLuminex AtheNA Multi-Lyte ANA test (Wampole Laboratories, Princeton,N.J.) was used to test for MAb reactivity to SSA/Ro, SS-B/La, Sm,ribonucleoprotein (RNP), Jo-1, double-stranded DNA (dsDNA), centromereB, and histone and was performed per the manufacturer's specificationsand as previously described (Haynes et al., Science 308, 1906-1908,2005). MAb concentrations assayed were 50, 25, 12.5 and 6.25 μg/ml. 10μl of each concentration were incubated with the luminex fluorescentbeads and the test performed per manufacturer's specifications.

Fluorescence-activated cell sorting (FACS) staining of cell-surfaceHIV-1 Env. FACS staining was performed as previously described(Chakrabarti et al., J Virol 85, 8217-8226, 2011; Koch et al., Virology313, 387-400, 2003). 48 hours following transfection, cells wereharvested and washed in FACS buffer (PBS, 5% HIFBS, 0.02% azide) andstained with monoclonal antibodies. The transfected cells were suspendedin FACS buffer and were incubated with the antibodies for one hour atroom temperature. The monoclonal antibody-cell mixture was washedextensively in FACS buffer and phycoerythrin (PE)-conjugated goatanti-human secondary antibody (Sigma) was added for one hour at a 1:200dilution, followed by extensive washing to remove unbound secondaryantibody. The antibody-PE-stained cells were acquired on a BD LSRIIinstrument and analyzed by FlowJo.

Antibody-virus washout experiments. From a starting concentration of 2mg/ml, 12.5 μl of 5-fold serially diluted antibodies in PBS were addedto 487.5 μl of DMEM containing 10% HIFCS and 15 μl of pseudovirus suchthat the final concentrations of antibodies were 50 μg/ml to 0.08 μg/mlin a total volume of 500 μl. In the “no inhibitor” control, the samevolume of PBS was added instead of antibody. The reaction mixture wasincubated for 30 minutes at 37° C. The 250 μl reaction mixture wasdiluted to 10 ml with complete DMEM, centrifuged at 25,000 rpm in a SW41rotor, for 2 hours at 4° C. The virus pellet was then washed twoadditional times with 10 ml of PBS. During the washing steps, thevirus-antibody complex was centrifuged at 40,000 rpm for 20 minutes at4° C. After the final wash, 250 μl of DMEM was added to the washed viruspellet and it was resuspended by gentle shaking at 4° C. for 30 min. 100μl of the suspended virus was used to infect 100 μl of TZM-b1 cells (0.2million/ml), in duplicate. From the remaining 250 μl of reactionmixture, an equal volume of the antibody virus mixture was used as a “nowashout” control. Plates were incubated at 37° C. in a CO₂ incubator for2 days. After 2 days, the luciferase assay was done as describedpreviously (Mascola et al. J Virol 76, 4810-4821, 2002). The data wasthen plotted to determine the neutralization mediated by the antibodiesin “wash” or “no wash” conditions

Structure determination and analysis. The antigen binding fragment of10E8 (Fab) was prepared using LysC digestion, as previously described(Ofek et al., Proc. Natl. Aca. Sci. U.S.A., 107, 17880-17887, 2010). TheIgG was first reduced with 100 mM DTT for one hour at 37° C., followedby one hour of dialysis in Hepes, pH 7.6, to reduce the DTTconcentration to 1 mM. Antibodies were then dialyzed against 2 mMiodoacetamide for 48 hours at 4° C., and subjected to a final dialysisagainst Hepes, pH 7.6, for 2 h. After reduction and alkylation,antibodies were cleaved with Lys-C (Roche), run over a Protein A columnto segregate away the Fc fragment, and then subjected to ion exchange(Mono S) and size-exclusion chromatography (S200). Purified 10E8 Fab wasincubated with 10-fold excess peptideRRR-NEQELLELDKWASLWNWFDITNWLWYIR(SEQ ID NO: 26)-RRR (American Peptide,CA) and the complex then set up set for 20° C. vapor diffusion sittingdrop crystallizations on the Honeybee 963 robot. 576 initial conditionsadapted from the commercially available Hampton (Hampton Research),Precipitant Synergy (Emerald Biosystems), and Wizard (EmeraldBiosystems) crystallization screens were set up and imaged using theRockimager (Formulatrix), followed by hand optimization of crystal hits.Crystals were grown in 40% PEG 400, 0.1 M NaCitrate, 0.1 M Tris pH 7.5diffracted to 2.1 Å resolution in a cryoprotectant composed of motherliquor supplemented with 15% 2R-3R-butanediol and excess peptide. Aftermounting the crystals on a loop, they were flash cooled and data wascollected at 1.00 Å wavelength at SER CAT ID-22 or BM-22 beamlines (APS)and processed using HKL-2000 (Otwinowski et al., MacromolecularCrystallography, Pt A 276, 307-326, 1997). Structures were solvedthrough molecular replacement with Phaser (McCoy et al., J ApplCrystallogr 40, 658-674, 2007; Winn et al., Acta Crystallogr D BiolCrystallogr 67, 235-242, 2011), using a previously obtained freestructure of 10E8 as a search model. Refinement of the structure wasundertaken with Phenix (Adams et al., Acta Crystallogr D BiolCrystallogr 58, 1948-1954, 2002), with iterative model building usingCoot (Emsley, P. & Cowtan, K. Acta Crystallogr D Biol Crystallogr 60,2126-2132 (2004). The structure was validated with MolProbity (Davis etal., Nucleic Acids Res 35, W375-383, 2007), yielding 97% and 99.8% ofresidues falling within most favored Ramachandran regions and allowedRamachandran regions, respectively. The structure was analyzed with APBS(Baker et al., Proceedings of the National Academy of Sciences of theUnited States of America 98, 10037-10041, 2001) for electrostatics(Ligplot; McDonald et al., J Mol Biol 238, 777-793, 1994), for directcontacts, PISA (Krissinel et al., J Mol Biol 372, 774-797, 2007), forburied surface areas, and LSQKAB (ccp4 Package; Winn, M. D. et al. ActaCrystallogr D Biol Crystallogr, 67, 235-242, 2011) for RMSD alignments.Helical wheels were generated using the program Pepwheel(150.185.138.86/cgi-bin/emboss/pepwheel). All graphics were preparedwith Pymol (PyMOL Molecular Graphics System).

Assessment of binding affinities of 10E8 and 10E8 variants to thegp41MPER. Surface-Plasmon Resonance (SPR) (BIACORE® T200, GE Healthcare)was used to assess binding affinity of wild type 10E8 to a gp41MPERpeptide. A biotinylated peptide composed of residues 656-683 of the gp41MPER (RRR-NEQELLELDKWASLWNWFDITNWLWYIR(SEQ ID NO: 26)-RRK-biotin;American Peptide, CA) was coupled to a BIACORE® SA chip to a surfacedensity of 20-50 Response Units (RU). The 10E8 fragment of antigenbinding (Fab) was then flowed over as analyte at concentrations rangingfrom 0.25 nM to 125 nM, at 2-fold serial dilutions, with association anddissociation phases of up to five minutes, at a flow rate of 30 ml/min.The binding of the 2F5 and 4E10 Fab controls to the same peptide wereexamined under identical conditions.

Binding affinities of the 10E8 paratope alanine mutants to the MPER werealso assessed with SPR, but using an antibody capture method. A BIACORE®CM5 chip was amine-coupled with anti-human Fc antibody to high surfacedensities of ˜10,000 RU. The 10E8 paratope variant IgGs were thencaptured to between 1500-2500 RU and a peptide composed of residues656-683 of the gp41MPER (RRR-NEQELLELDKWASLWNWFDITNWLWYIR(SEQ ID NO:26)-RRR) flowed over as analyte at 2-fold serial dilutions starting at500 nM (with the exception of HC D30A, W100bA, S100cA, P100fA, whichstarted at 250 nM). Association and dissociation phases spanned threeminutes and five minutes, respectively, at a flow rate of 30 μl/min.Binding sensograms were fit with 1:1 Langmuir models using BIACORE®BiaEvaluation® Software (GE Healthcare). In all cases, BIACORE® HBSEP+buffer was used (10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.1%P-20).

PCR amplification and sequencing. Extraction of viral RNA from plasmaand cDNA synthesis were performed as previously described (Imamichi etal., J Infect Dis 183, 36-50, 2001). Single molecules of a 588 byfragment, encompassing the MPER region of the HIV-1 envelope gene,obtained through limiting dilution, were PCR-amplified with the ExpandHigh Fidelity PCR System (Roche Applied Science, Indianapolis, Ind.)using the following primer sets: +7789 (sense)5′-TCTTAGGAGCAGCAGGAAGCACTATGGG-3′ (SEQ ID NO: 193) and -8524(antisense) 5′-GTAAGTCTCTCAAGCGGTGGTAGC-3′ (SEQ ID NO: 194) in a firstround reaction; +7850 (sense) 5′-ACAATTATTGTCTGGTATAGTGCAACAGCA-3′ (SEQID NO: 195) and -8413 (antisense) 5′-CCACCTTCTTCTTCGATTCCTTCGG-3′ (SEQID NO: 196) in a second round reaction. Each round of PCR consisted of25 cycles, with the initial denaturation at 94° C. for two minutes,followed by 25 cycles of denaturation at 94° C. for 15 seconds,annealing at 50° C. for 30 seconds, and extension at 72° C. for 1minute, with the final extension at 72° C. for 7 min. The PCR productswere purified with the QIA quick PCR purification kit (QIAGEN, Valencia,Calif.), and then cloned into pCR2.1-TOPO vector (TOPO TA Cloning it,Invitrogen, Carlsbad, Calif.) for sequence analysis of individualmolecular clones. The DNAs from 18 independent clones were sequencedwith the ABI BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit(Applied Biosystems, Foster City, Calif.) and analyzed with the ABIPRISM 3130x1 Genetic Analyzer (Applied Biosystems, Foster City, Calif.).

Statistical analysis. The relationship between the potency of N152patient serum and 10E8, and the relationship between 10E8 variantbinding and neutralization were evaluated by the Spearman rank method.

Deposits. The nucleotide sequence of 10E8 heavy and light chains havebeen submitted to GenBank under accession numbers JX645769 and JX645770,each of which is incorporated by reference herein as present in GenBankon Sep. 18, 2012. Coordinates and structure factors for 10E8 Fab incomplex with the gp41MPER have been deposited with the Protein Data Bankunder accession code 4G6F which is incorporated by reference herein aspresent in GenBank on Sep. 18, 2012.

Example 2 Neutralization Assays

This examples describes a method for testing of the neutralizationbreadth and potency of the antibodies disclosed herein.

Neutralization of the monoclonal antibodies was measured using a singleround infection by HIV-1 Env-pseudoviruses and TZM-b1 target cells.HIV-1 Env-pseudoviruses were generated by co-transfection of 293T cellswith pSG3ΔEnv backbone containing a luciferase reporter gene and asecond plasmid that expressed HIV-1 Env. At 72 hours post-transfection,supernatant containing pseudoviruses were harvested and frozen at −80°C. until further use. Anti-gp41 membrane-proximal external region (MPER)specific activity of patient serum and antibodies was measured using theHIV-2/HIV-1 MPER chimeras. Wide-type HIV-2 7312A was used as a control.The 50% inhibitory concentration (IC50) was calculated as theantibody/inhibitor concentrations causing a 50% reduction of infection.For the competition assay, a fixed concentration of peptide wasincubated with serially diluted 2F5, 4E10, Z13E1 or 10E8 Ab at 37° C.for 30 minutes before incubation of 7312A-C1 chimera. Epitope mappingassay was assessed by adding 0.5 μg/ml 10E8 Ab to serial dilutions of4E10 peptide or its alanine mutants at 37° C. for 30 minutes prior tothe addition of 7312A-C1 chimera. The neutralization blocking effect ofthe peptides was calculated as the fold change in the IC50 value of theantibody in the presence of 4E10 alanine mutants compared to the 4E10wild-type peptide. Neutralization of 10E8 against HIV-1 COT6.15 alaninemutant pseudoviruses was also measured using a TZM-b1 assay (FIG. 36).Further neutralization of several HIV-1 strains was tested with 10E8antibody as well as antibodies containing cross-complemented 10E8, 7H6,and 7N16 heavy and light chains (FIG. 37).

Example 3 ELISA Assays

Each antigen at 2 μg/ml was used to coat 96-well plates overnight at 4°C. Coated plates were blocked with BLOTTO buffer (PBS, 1% FBS, 5%non-fat milk) for one hour at room temperature, followed by incubationwith antibody serially diluted in disruption buffer (PBS, 5% FBS, 2%BSA, 1% Tween-20) for one hour at room temperature. Horseradishperoxidase (HRP)-conjugated goat anti-human IgG antibody at 1:10,000 wasadded for one hour at room temperature. Plates were washed between eachstep with 0.2% Tween 20 in PBS. Plates were developed using3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma) and read at 450 nm.

Example 4 HIV-1 Monoclonal Neutralizing Antibodies Specific to Gp41 forDetecting HIV-1 in a Sample or a Subject

This example describes the use of HIV-1 monoclonal neutralizingantibodies specific to gp41 for the detection of HIV-1 in a sample or asubject. This example further describes the use of these antibodies toconfirm the diagnosis of HIV-1 in a subject.

A biological sample, such as a blood sample, is obtained from thepatient diagnosed with, undergoing screening for, or suspected of havingan HIV-1 infection. A blood sample taken from a patient who is notinfected is used as a control, although a standard result can also beused as a control. An ELISA is performed to detect the presence of HIV-1in the blood sample. Proteins present in the blood samples (the patientsample and control sample) are immobilized on a solid support, such as a96-well plate, according to methods well known in the art (see, forexample, Robinson et al., Lancet 362:1612-1616, 2003, incorporatedherein by reference). Following immobilization, HIV-1 monoclonalneutralizing antibodies specific to gp41 that are directly labeled witha fluorescent marker are applied to the protein-immobilized plate. Theplate is washed in an appropriate buffer, such as PBS, to remove anyunbound antibody and to minimize non-specific binding of antibody.Fluorescence can be detected using a fluorometric plate reader accordingto standard methods. An increase in fluorescence intensity of thepatient sample, relative to the control sample, indicates the gp41antibody specifically bound proteins from the blood sample, thusdetecting the presence of HIV-1 protein in the sample. Detection ofHIV-1 protein in the patient sample indicates the patient has HIV-1, orconfirms diagnosis of HIV-1 in the subject.

Example 5 HIV-1 Monoclonal Neutralizing Antibodies Specific for Gp41 forthe Treatment of HIV-1

This example describes a particular method that can be used to treat HIVin a human subject by administration of one or more gp41-specific humanneutralizing mAbs. Although particular methods, dosages, and modes ofadministrations are provided, one skilled in the art will appreciatethat variations can be made without substantially affecting thetreatment.

Based upon the teaching disclosed herein, HIV-1 can be treated byadministering a therapeutically effective amount of one or more of theneutralizing mAbs described herein, thereby reducing or eliminating HIVinfection.

Screening subjects. In particular examples, the subject is firstscreened to determine if they have an HIV infection. Examples of methodsthat can be used to screen for HIV infection include a combination ofmeasuring a subject's CD4+ T cell count and the level of HIV in serumblood levels. Additional methods using the gp41-specific mAbs describedherein can also be used to screen for HIV.

In some examples, HIV testing consists of initial screening with anenzyme-linked immunosorbent assay (ELISA) to detect antibodies to HIV,such as to HIV-1. Specimens with a nonreactive result from the initialELISA are considered HIV-negative unless new exposure to an infectedpartner or partner of unknown HIV status has occurred. Specimens with areactive ELISA result are retested in duplicate. If the result of eitherduplicate test is reactive, the specimen is reported as repeatedlyreactive and undergoes confirmatory testing with a more specificsupplemental test (e.g., Western blot or an immunofluorescence assay(IFA)). Specimens that are repeatedly reactive by ELISA and positive byIFA or reactive by Western blot are considered HIV-positive andindicative of HIV infection. Specimens that are repeatedlyELISA-reactive occasionally provide an indeterminate Western blotresult, which may be either an incomplete antibody response to HIV in aninfected person, or nonspecific reactions in an uninfected person. IFAcan be used to confirm infection in these ambiguous cases. In someinstances, a second specimen will be collected more than a month laterand retested for subjects with indeterminate Western blot results. Inadditional examples, nucleic acid testing (e.g., viral RNA or proviralDNA amplification method) can also help diagnosis in certain situations.

The detection of HIV in a subject's blood is indicative that the subjectis infected with HIV and is a candidate for receiving the therapeuticcompositions disclosed herein. Moreover, detection of a CD4+ T cellcount below 350 per microliter, such as 200 cells per microliter, isalso indicative that the subject is likely to have an HIV infection.

Pre-screening is not required prior to administration of the therapeuticcompositions disclosed herein.

Pre-treatment of subjects. In particular examples, the subject istreated prior to administration of a therapeutic agent that includes oneor more antiretroviral therapies known to those of skill in the art.However, such pre-treatment is not always required, and can bedetermined by a skilled clinician.

Administration of therapeutic compositions. Following subject selection,a therapeutically effective dose of a gp41-specific neutralizing mAbdescribed herein is administered to the subject (such as an adult humanor a newborn infant either at risk for contracting HIV or known to beinfected with HIV). Additional agents, such as anti-viral agents, canalso be administered to the subject simultaneously or prior to orfollowing administration of the disclosed agents. Administration can beachieved by any method known in the art, such as oral administration,inhalation, intravenous, intramuscular, intraperitoneal, orsubcutaneous.

The amount of the composition administered to prevent, reduce, inhibit,and/or treat HIV or a condition associated with it depends on thesubject being treated, the severity of the disorder, and the manner ofadministration of the therapeutic composition. Ideally, atherapeutically effective amount of an agent is the amount sufficient toprevent, reduce, and/or inhibit, and/or treat the condition (e.g., HIV)in a subject without causing a substantial cytotoxic effect in thesubject. An effective amount can be readily determined by one skilled inthe art, for example using routine trials establishing dose responsecurves. As such, these compositions may be formulated with an inertdiluent or with an pharmaceutically acceptable carrier.

In one specific example, antibodies are administered at 5 mg per kgevery two weeks or 10 mg per kg every two weeks depending upon theparticular stage of HIV. In an example, the antibodies are administeredcontinuously. In another example, antibodies or antibody fragments areadministered at 50 μg per kg given twice a week for 2 to 3 weeks.

Administration of the therapeutic compositions can be taken long term(for example over a period of months or years).

Assessment. Following the administration of one or more therapies,subjects with HIV can be monitored for reductions in HIV levels,increases in a subject's CD4+ T cell count, or reductions in one or moreclinical symptoms associated with HIV. In particular examples, subjectsare analyzed one or more times, starting 7 days following treatment.Subjects can be monitored using any method known in the art. Forexample, biological samples from the subject, including blood, can beobtained and alterations in HIV or CD4+ T cell levels evaluated.

Additional treatments. In particular examples, if subjects are stable orhave a minor, mixed or partial response to treatment, they can bere-treated after re-evaluation with the same schedule and preparation ofagents that they previously received for the desired amount of time,including the duration of a subject's lifetime. A partial response is areduction, such as at least a 10%, at least 20%, at least 30%, at least40%, at least 50%, or at least 70% in HIV infection, HIV replication orcombination thereof. A partial response may also be an increase in CD4+T cell count such as at least 350 T cells per microliter.

Example 6 PCR for 454 Sequencing of Patient N152 Cells

This example described a PCR assay for amplifying sample DNA for deepsequencing. Process included generation of cDNA from patient cells usingRT PCR followed by amplification of VH3 and VL3 genes from the generatedcDNA.

-   Sample of patient N152 had 33-36 million PBMCs/ml.-   mRNA was prepared using Qiagen Oligotex Kit (as described in    manufacturer's instructions).-   RT PCR was performed on mRNA using Invitrogen reagents (which    included oligo dT as primer, and Superscript II RT).    After RT PCR reactions, cDNA was then subjected to amplification    using VH3 and VL3 gene specific primers meant to amplify from the    leader sequences of the alleles IGHV3-15*05 and IGLV3-19*01, which    are the putative precursors of 10E8.    The primers used were as follows:

5′ Heavy Chain >XLR-A_5L-VH3 (SEQ ID NO: 28)CCATCTCATCCCTGCGTGTCTCCGACTCAGAAGGTGTCCAGTGTGARGTG CAG >XLR-A_VH3-L1-MP(SEQ ID NO: 29) CCATCTCATCCCTGCGTGTCTCCGACTCAGGCTATTTTAAAAGG-TGTCCAATGT >XLR-A_VH3/4_L1_MP (SEQ ID NO: 30)CCATCTCATCCCTGCGTGTCTCCGACTCAGGTGGCAGCTCCCAGATG-GGTCCTGTC >XLR-A_VH3/4_L3_MP (SEQ ID NO: 31)CCATCTCATCCCTGCGTGTCTCCGACTCAGGTTGCAGTTTTAAA- AGGTGTCCAGTG 5′Light Chain >XLR-A_5L-VL3 (SEQ ID NO: 32)CCATCTCATCCCTGCGTGTCTCCGACTCAGGCTCTGTGACCTCCTATGAG CTG >XLR-A_5MP-VL3-1(SEQ ID NO: 33) CCATCTCATCCCTGCGTGTCTCCGACTCAGGCTTACTGCACA-GGATCCGTGGCC >XLR-A_5MP-VL3-19 (SEQ ID NO: 34)CCATCTCATCCCTGCGTGTCTCCGACTCAGACTCTTTGCAT-AGGTTCTGTGGTT >XLR-A_5MP-VL3-21 (SEQ ID NO: 35)CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTCACTGCACAGGC- TCTGTGACCSamples were gel extracted and purified by phenol-chloroform.

-   Final yield: 1.15 μg Heavy Chain; 0.9 μl Light Chain-   0.5 μg of each chain was sent for 454 sequencing.    Total number of sequences obtained from a full 454 chip for each    chain: Heavy Chain: 843084 raw sequences, 37669 sequences belonging    to IGHV3-15 family; Light Chain: 1219214 raw sequences, 91951    sequences belonging to IGKV3-15 family

Example 7 Method for Isolating and Producing Monoclonal Antibodieswithout Prior Knowledge of Antigen Specificity

This example illustrates a method to isolate and produce all antibodiesthat an organism produces against a target antigen, such as a virions,cancers, or toxins. Current methods for the isolation and production ofmonoclonal antibodies rely upon specific, known epitopes to isolate andproduce new monoclonal antibodies. Using these methods, B cells aresorted and immunoglobulin genes are isolated based on epitopespecificity. These methodologies, however, may be biased because theyrely upon previously known epitopes or antibody specificities.Additionally, because these methodologies rely upon previously knownantibody specificities or epitopes, these methodologies do not permitthe discovery of new monoclonal antibodies with unknown epitopespecificities. Unlike current methodologies, this methodology startswith a population of B-cells from an organism which is representative ofall B cells produced by the organism. This population is expanded andscreened for activity against a functionality or antigen of interestsuch as a virion, toxin, protein, or cancer. This permits the isolationof a full repertoire of memory B-cells that an organism producesspecific to an antigen without prior knowledge of specificity or bindingcharacteristics. For example, memory B-cells in this repertoire can bescreened for a functional activity (such as neutralization activity) andused to produce monoclonal antibodies specific to the antigen.

Methods

Staining and single-cell sorting of memory B cells are performed asfollows.

Isolation of B Cells

PBMCs from a subject previously exposed to the target antigen (such as asubject with HIV-1 infection, if the target antigen is an HIV-1 antigen)are stained with antibody cocktail consisting of anti-CD19-PE-Cy7 (BDBioscience), IgA-APC (Jackson ImmunoResearch Laboratories Inc.),IgD-FITC (BD Pharmingen), and IgM-PE (Jackson ImmunoResearchLaboratories Inc.) at 4° C. in dark for 30 min. The cells are thenwashed with 10 ml PBS-BSA buffer and resuspended in 500 μl PBS-BSA.CD19+IgA-IgD-IgM- memory B cells are sorted using a FACSAria III cellsorter (Becton Dickinson). FIG. 42 illustrates results of FACS isolationof CD19⁺IgA⁻IgD⁻IgM⁻ B cells from a PBMC sample.

Additional Description of Isolation of B Cells

-   -   1. Prepare FACSARIA III® cell sorter (Becton Dickinson, Franklin        Lakes, N.J.):        -   a. turn on, warm up, and run CST; run Auto Drop delay            (follow steps in manual);        -   b. make a bottle of sterile PBS-BSA (or re-filter existing            bottle)        -   c. sterilize the tubing: (1) load a sample of 15% contrad            and run for five minutes at flow rate 8; (2) load a sample            of 10% bleach and run for five minutes at flow rate 8; (3)            load a sample of sterile PBS-BSA (in sterile tube) and run            for minutes at flow rate 8; (4) reset to flow rate 1        -   d. aim the sort stream: (1) put an empty guava tube in sort            block and attach the block; (2) open stream door; (3) Diva:            in the sidestream window (called “70 micron”), open waste            drawer; make sure sliders 1, 3, 4 are set to zero, slider 2            should be around 48; (4) click Voltage then Test Sort; (5)            look in the stream door—is the sort stream hitting the            center of the tube?; (6) use sliders on sidestream window to            adjust stream so it's centered; (7) click Voltage to turn            off; close waste drawer        -   e. DIVA (Aria software): copy an old template by choosing a            previous sort, then “Duplicate without data” or set up new            experiment with gating as shown below        -   f. Attach chiller tubing to sort block; turn on chiller    -   2. Prepare cells:        -   a. thoroughly clean hood and pipets with ethanol before            starting        -   b. make at least 400 ml IMDM(with glutamax)/10% FBS/mycozap        -   c. warm two 15 ml conical tube of 7.5 ml IMDM/10% FBS with            15 μl benzonase        -   d. make staining master mix in eppindorf tube:            -   spin IgM and IgA antibodies 1 min before aliquotting            -   make master mix: 50 μl per 50 million cells antibody

for 1x for 2.5x CD19-PE-Cy7 0.5 μl 1.25 μl IgM-PE 1.0 μl  2.5 μl IgA-APC2.5 μl 6.25 μl IgD-FITC 2.5 μl 6.25 μl PBS-BSA 43.5 μl  108.75 μl 

-   -   spin 20 minutes at 4° C. in microfuge; FACS Dyes (PE:        Phycoerythrin; Cy7: a cyanine dye; APC: Allophycocyanin; FITC:        fluorescein isothiocyanate; CD19-PE-Cy7 (anti-CD19 mAb        conjugated with Dyes—specific to B-cells (except plasma b        cells); IgA: anti IgA mAb; IgD: anti-IgD mAb; IgM: anti-IgM mAb        -   e. thaw 2 vials patient cells: (1) warm vial in water bath            until floating ice is visible; (2) add 1 ml prewarmed            medium/benzonase to vial, let sit 15 sec.; (3) move all vial            contents to the 15 ml conical tube of warm medium; (4)            pellet 1200 rpm 10 min; (5) resuspended each in 1 ml            PBS-BSA, combine, move 50 μl to each of 5 conical tubes; (6)            pellet all 6 conical tubes; (7) NOTE—if only one vial            needed, you can skip steps v. and vi.        -   f. resuspended cell pellets: (1) main sample: in 100 μl            master mix; (2) comps in 50 μl PBS-BSA, add single stains            (0.5 μl CD19-PE-Cy7 etc)        -   g. 30 min 4° C. covered in foil        -   h. wash: add 1000 μl PBS-BSA with P1000, mix well, add 2 ml            more PBS-BSA        -   i. pellet, meanwhile open new bag of filter cap tubes in            hood and label them        -   j. resuspended in 500 μl PBS-BSA and transfer to sterile            filter-cap tubes by squirting cells thru the cap (touch tip            to cap while squirting)        -   k. keep cold until ready to flow    -   3. Sort cells: (1) load on Aria, gate as shown in example; use        old compensation if voltages look OK, otherwise, adjust voltages        and run compensation; (2) adjust flow rate between 1-2 so that        event rate is as high as possible but no more than 13,000        evt/sec. monitor this during sorts; (3) set up sort (Click on        Sort >New sort layout; Purity 1.5; Sort: continuous; Choose gate        P6 for sorting into on Left); (4) Put 250 μl medium into 2 ml        o-ring tube, load into sort block e. Test sort 500 cells; (5)        Unload sample, return to cold; (6) Run sample line backflush for        1-2 minutes, then load fresh tube of PBS-BSA at flow; (7) rate 8        for two minutes to clean out main sample; (8) Remove sort tube        from sort block, use P1000 to gently wash down the sides using        medium in tube; (9) Move all to a flow tube; (10) Flow and        record the post sort purity; (12) Load main sample on Aria and        sort 20000-30000 B cells; (13) Repeat steps 5-7; (14) Take 10        μl, add to 100 μl PBS-BSA in flow tube, flow and record the post        sort purity; (15) Put 270 μl Guava ViaCount in a guava tube, add        30 μl sorted cells, two minutes, then count on Guava (record        concentration only; it will not accept 0.25 ml as volume); (16)        Calculate amount of cells needed to plate at desired density.        Treating the Cells with Growth Factors to Induce Cell Division

CD19+IgA-IgD-IgM- Memory B cells are resuspended in Iscove's ModifiedDulbecco's Medium IMDM medium with 10% FBS containing 100 U/ml IL-2, 50ng/ml IL-21 and 1×10⁵/ml irradiated 3T3-msCD40L feeder cells produced aspreviously described (Kershaw et al., Cancer Res., 61: 7920-7924, 2001).B cells are seeded into 384-well microtiter plates at a density of 4cells/well in a final volume of 50

Additional Description of Treating Cells with Growth Factors

-   -   1. Prepare plating conditions: (1) Thaw 1 vial of 35×10⁶        irradiated 3T3-msCD40L cells (benzonase method), resuspended in        10 ml IMDM/10% FBS; (2) Calculate reagent amounts        -   a. Concentrations (Feeder Mix): 3T3-msCD40L: 5000 cells/50            ul/well=10⁵ cells/ml; IL2 100 u/ml; IL21 50 u/ml; In IMDM            cell culture media/10% FBS (see above); For 20 plates=350            ml: 336 ml IMDM/10% FBS; 3.5 ml IL2 (10000 u/ml); 175 μl            IL21; 10 ml 3T3 msCD40L        -   b. Make feeder mix as calculated; For 10 plates: set aside            12.5 ml for no-B cell control.    -   2. Plate cells: (1) Use 384-well white plates; label them on lid        and on side of plate; (2) Use 12-channel pipets; (3) Angled        plate holders may be useful; (4) Place 100 μl sterile dI-H2O        with gent in outer wells (need 7.7 ml/plate); (5) Before adding        b cells to mix, plate 50 μl/well feeders mix in row D of all        plates (this is; (6) no-antibody control); (7) Add appropriate        amount of B cells to remaining feeders mix (For 10 plates, 2.5 B        cells/well=50 cells/ml, need 8125 B cells); (8) Move cell mix to        large sterile basins, and plate 50 μl/well in inner 308 wells        (except row D); (9) Be sure to mix the cells in the basin        frequently; (10) Plate an extra quarter-plate for ELISA        later; (11) Move to back of incubator, leave undisturbed for 13        days    -   3. Supplies: IL2—Roche-11147528001 (50 ml);        IL21—Invitrogen—PHC0215 (25 ug);        IMDM+Glutamax—Invitrogen—31980-097 (1×10 btls)        Incubation and Supernatant Neutralization Screening

After 13 days of incubation, 40 μl of culture supernatants from eachwell are collected and screened for neutralization activity using afunctional target of the antigen of interest (for example, if an HIV-1antigen is the target antigen, the neutralization assay can be a highthroughput micro-neutralization assay against HIV-1MN.03 andHIV-1Bal.26).

Additional Description of Collection of Supernatant for NeutralizationAssay

-   -   1. Collection of supernatant for Neutralization assay        -   a. Prepare an ELISA plate beforehand (coat a Maxisorp™ plate            with anti-IgG); Clean hood and pipets with ethanol and then            RNaseAway™; label as many 384 well white plates as you have            day 11 plates        -   b. move 40 μl from each well of day 11 plate to            corresponding well of fresh plate: (1) use a 12 channel            pipette; (2) get tip as far down in well as possible—Ok to            touch bottom lightly, move tip up slightly, then pull            up; (3) dispense to new plate; (4) cover sups plates with            foil sticker and put lid on; quarter-plate goes to 4°            C.; (5) store sups plates at −80 and use as needed for            neutralization assay        -   c. make catch-lysis buffer—enough for 308 wells*1.15*number            of plates: for 1 well (0.3 μl Tris, pH 8, 1M; 0.25 μl RNase            Inhibitor; 19.45 μl depc-treated H2O); put 20 μl catch-lysis            buffer onto all B cells wells (dispense with multi-channel            pipette; cover with foil sticker and put lid on; store at            −80° C.); perform ELISA using 10 μl of sup from            quarter-plate to measure IgG concentration and hit rate            Production of Monoclonal Antibodies

B cells in each well are lysed with 20 μl lysis buffer containing 0.25μl of RNase inhibitor (New England Biolabs Inc.), 0.3 μl of 1M Tris pH8(Quality Biological Inc.) and 19.45 μl DEPC-treated H2O. The plates withB cells are stored at −80° C. The variable region of the heavy chain andthe light chain of the immunoglobulin gene are amplified by RT-PCR fromthe wells that scored positive in the neutralization assay. The cDNAproduct is used as template in the PCR reaction. In order to amplify thehighly somatically mutated immunoglobulin gene, two sets of primers asdescribed previously (Tiller et al., J. Immunological Methods,329:112-124, 2008) are used in two independent PCRs. One set of primersincludes the forward primers and the reverse primers specific for theleader region and constant region of IgH, Igκ or Igλ, respectively. Theother set of primers includes the forward primer mixes specific for FWR1and respective reverse primers specific for the IgH, Igκ and Igλ Jgenes. All PCRs are performed in 96-well PCR plates in a total volume of50 μl containing 20 nM each primer or primer mix, 10 nM each dNTP(Invitrogen), 10 μl 5× Q-solution (Qiagen) and 1.2 U HotStar Taq DNApolymerase (Qiagen). From the positive PCR reactions, pools of the VH orVL-region DNA are ligated to a pCR2.1-Topo-TA vector (Invitrogen) forsequencing before cloning into the corresponding Igγ1, Igκ and Igλexpression vector. 10 μg of heavy and light chain plasmids, cloned fromthe same well and combined in all possible heavy and light chain pairs,is mixed with 40 μl FUGENE® 6 (Roche) in 1500 μl DMEM (Gibco) andco-transfected into 293T cells. The full-length IgG is purified using arecombinant protein-A column (GE Healthcare).

Neutralization Assays for Monoclonal Antibodies

Following purification of full length IgG, the produced antibodies aretested for neutralization activity.

For example, if the antigen of interest is an HIV-1 antigen, theneutralization activity of the monoclonal antibodies can be measuredusing single-round HIV-1 Env-pseudoviruses infection of TZM-b1 cells.HIV-1 Env-pseudoviruses can be generated by co-transfection of 293Tcells with pSG3 Env backbone and a second plasmid that expressed HIV-1Env. At 72 hours post-transfection, supernatants containing pseudovirusare harvested and frozen at −80° C. until further use. In theneutralization assay, 10 μl of 5-fold serially diluted patient serum ormAb is incubated with 40 μl pseudovirus in a 96-well plate at 37° C. for30 minutes before addition of TZM-b1 cells. After 2 days of incubation,cells are lysed and the viral infectivity quantified by measuringluciferase activity with a VICTOR® Light luminometer (Perkin Elmer). The50% inhibitory concentration (IC50) is calculated as the antibodyconcentration that reduces infection by 50%.

Example 8 10E8 Modifications

This example illustrates modifications of the 10E8 antibody to increaseaffinity for gp120, without increasing autoreactivity. As describedbelow, several approaches were undertaken to identify and designimprovements to the 10E8 antibody, including Structure-based alaninescanning mutagenesis of 10E8 paratope; Structure-based mutagenesis toenhance hydrophobic interactions; Partial germline reversion;combination of these approaches, and 454 deep pyrosequencing of patientN152 B-cells to identify phylogenetic variants of 10E8 with improvedpotency. In addition, combinations of these four approaches were alsoundertaken, combining the best elements and results from each. Below isa summary of each of the four approaches with the rationale behind each.

(1) Structure-Based Alanine Scanning Mutagenesis of 10E8 Paratope

Based on the crystal structure of 10E8 Fab with the gp41MPER (describedabove), heavy chain residues of the paratope of 10E8 were mutated toalanine. Mutant antibody constructs were generated using standardtechniques, and the antibodies were expressed and purified as describedin Example 1. All residues of the 10E8 paratope were mutated to alanine(see FIG. 43), and several residue positions led to enhanced10E8-mediated virus neutralization (see FIGS. 32 and 49). These include10E8 residues D28, D30, N31, T52, E53, S56, D58, and Y100e (Kabatnumbering). Neutralization assays were performed as described in Example2.

(2) Structure-Based Mutagenesis to Enhance Hydrophobic Interactions

Based on the crystal structure of 10E8 Fab with the gp41MPER, residuesthat were deemed to be co-planar with the 10E8 epitope were mutated tohydrophobic residues (see FIGS. 44A and 44B). Mutant antibody constructswere generated using standard techniques, and the antibodies wereexpressed and purified as described in Example 1. As shown in FIG. 45,several residue positions, both singly and in combination, resulted inincreased neutralization potency of 10E8. Of the mutants listed in FIG.45, mutation of residue S74 to tryptophan generated the greatestincrease in neutralization, followed by the double mutant D30-N31 andthe single mutant D28. Neutralization assays were performed as describedin Example 2.

(3) Partial Germline Reversion

CDR grafting was used to revert a fraction of the 10E8 maturationchanges back to the germline sequence. CDR grafting is a procedure thatis typically used for humanization of antibodies from a non-human (e.g.,mouse) source: the procedure uses a sequence alignment of the non-humanantibody with a human counterpart (typically a human germline gene) andretains the original antibody CDR's but mutates selected frameworkresidues to the respective human identities. Framework residues areselected for mutation based on structural and sequence analysis. Incertain instances, not all of the observed maturation changes in anantibody may be beneficial, and partial reversion to germline may helpimprove certain antibody properties, such as increased half-life,decreased immunogenicity, or improved potency. By applying CDR graftingto 10E8, three different heavy chain revertants and three differentlight partially reverted chains were designed (see FIG. 47). At theamino acid level, the mutation rates were decreased from 28% to 12-20%for the heavy chain and from 21% to 7-14% for the light chain. Themutated antibodies were constructed and produced using standard methodsas described in Example 1. Different combinations of heavy and lightchain revertants were tested for binding to MPER and for neutralization(see FIG. 48). The largest improvement in neutralization (<2-fold) wasobserved for the revertant that included the heavy and light chainreversions closest to the mature antibody (“10E8-R3,” which includes the10E8gH03 heavy chain (SEQ ID NO: 149) and the 10E8gL03 light chain (SEQID NO: 152)). Additional improvements were observed when the revertantswere paired with heavy/light chains from other sources (such as deepsequencing, discussed below).

(4) Additional Mutations

Based on neutralization data, revertant analysis, paratope alaninescanning, and enhanced hydrophobicity designs described above, thefollowing additional mutations were introduced into the 10E8gH03 heavychain variant of 10E8 and the 10E8gL03 light chain variant of 10E8,antibodies were constructed and produced according to standard methodsas described in Example 1, and tested for neutralization activityaccording to methods described in Example 2 (FIG. 49)

TABLE 1 Additional 10E8 variant heavy and light chains Heavy chain LightChain 10E8gH03 (SEQ 10E8gL03 (SEQ ID NO: 149) ID NO: 152) withAdditional with Additional Name in FIG. 51 mutants” mutants 10E8H3_1F +L3 1F — 10E8H3_1L + L3 1L — 10E8H3_1W + L3 1W — 10E8H3_A32Q + L3 A32Q —10E8H3_D30AE56A + L3 D30A + E53A — 10E8H3_D30AE56AS77W + L3 D30A + —E53A + S74W 10E8H3_D30WN31F + L3 D30W + N31F — 10E8H3_D30WN31FE56A + L3D30W + — N31F + E53A 10E8H3_E56A + L3 E53A — 10E8H3_E56F + L3 E53F —10E8H3_E56L + L3 E53L — 10E8H3_E56M + L3 E53M — 10E8H3_E56V + L3 E53V —10E8H3_E56W + L3 1E53W — 10E8H3_N31F + L3 10N31F — 10E8H3_N31Y + L3 N31Y— 10E8H3_S59H + L3 S56H — 10E8H3_S77F + L3 S74F — 10E8H3_S77L + L3 S74L— 10E8H3_S77M + L3 S74M — 10E8H3_S77W + L3 S74W — 10E8L3_2L + H3 — 2L10E8L3_3W + H3 — 3W 10E8L3_G94W + H3 — G94W 10E8L3_S93W + H3 — S93W(5) 454 Deep Pyrosequencing to Identify Phylogenetic Variants of 10E8with Improved Potency

This example illustrates the identification and functional pairing of10E8-like heavy and light chains sequences determined in separate 454pyrosequencing reactions. Beginning from the wild-type sequence of 10E8neutralizing antibody, clonal variants for heavy and light chain wereidentified and assessed for function by pairing with the wild-type 10E8complementary chain. The phylogenetic trees of the heavy and lightchains revealed similar branch topologies around the wild-type 10E8sequences, allowing branches of the heavy- and light-chain phylogenetictrees to be matched based on their relative distances from 10E8. Byassessing a matrix of antibodies reconstituted from matched andmismatched branches for neutralization of HIV-1 and reactivity withauto-antigens, the impact of phylogenetic pairing on function wasquantified.

The broadly neutralizing antibody 10E8 was identified in theHIV-1-infected donor N152, and recognizes a helix in themembrane-proximal external region (MPER) just prior to thetransmembrane-spanning region of the HIV-1 gp41 glycoprotein, andneutralizes 98% of diverse HIV-1 isolates at a 50% inhibitoryconcentration (IC50) of 0.32 ug/ml. The heavy chain of antibody 10E8derives from IgHV3-15 and IgHJ1, has a third complementarity determiningregion (CDR H3) of 22 amino acids, and displays a somatic mutation rateof 21%. The light chain of antibody 10E8 derives from IgVL3-19 andIgLJ3, has a CDR L3 of 12 amino acids, and displays a somatic mutationrate of 14%. Deep sequencing of donor B cell transcripts usingpolymerase chain reaction (PCR) to amplify IgG heavy chain sequencesfrom the IgHV3 family and to amplify IgG light chain sequences from theIgVL3 family was performed as described in Example 6. mRNA from anestimated 5 million peripheral blood mononuclear cells (PBMCs) was usedfor reverse transcription to produce template cDNA, and, in both cases,primers that were upstream from the start of the V-gene leader sequencesand downstream from the end of J chain were used.

Roche 454 pyrosequencing provided 843,084 heavy chain reads and1,219,214 light chain reads for donor N152 (see FIG. 50). After primaryanalysis using a bioinformatics pipeline 36,318 heavy chain sequenceswere assigned to the IgHV1-3 allelic family and 54,583 light chainsequences were assigned to the IgVL3-IgJ3 allelic families (see, e.g.,Wu et al., Science, 333, 1593-1602, 2011, and Zhu et al., Frontiers inmicrobiology, 3, 315-315, 2012). Reads were analyzed for identity to10E8 and divergence from the unmutated V-genes, and their frequenciesplotted on identity/divergence grids (FIGS. 50A and 50B, left panels).Notably with the heavy chain several well-separated islands of highidentify and about 25% divergence were observed, and with the lightchain, a single well-separated island of high identify and about 15%divergence was observed. Grid-based sampling of the highidentity-divergence region, selected 61 heavy chain sequences and 48light chain sequences. The phylogenetic relationship of these sequencesto 10E8 was analyzed (FIGS. 50C and 50D) and also synthesized,reconstituted with the complementary wild-type 10E8 chain, and expressedby transient transfection in a 96-well format. Enzyme-linkedimmunosorbent assays (ELISAs) of the expressed 10E8 variants identified11 heavy chains and 24 light chains (the nomenclature and sequences ofthese heavy and light chains is shown in FIG. 51 and FIG. 59) which whenpaired with the partner 10E8 chains bound to a peptide corresponding tothe entire MPER of HIV-1 gp41 (FIGS. 50A and 50B, right panels). On a6-HIV-1-isolate panel, up to ˜5-fold increases in neutralization potencywere observed (FIGS. 50E and 50F).

The functional 10E8 heavy chains derived from three distinct islands inthe identity/divergence plots (FIG. 50A), and exhibited sequences andmutational patterns consistent with a common clonal origin (FIG. 51A).Mutations clustered in CDR H1 and H3, and also in the first, third andfourth framework regions (FR1, FR3 and FR4). The most divergentsequence, gVRC-H11_(dN152), had 25 amino acid changes, corresponding to19.1% difference from the wild-type 10E8 heavy chain. The functional10E8 light chains derived from several regions of theidentity/divergence plots, including a single distinct island andseveral regions overlapping the primary light chain population (FIG.50B). Like heavy chain, the functional light chains exhibited mutationalpatterns consistent with a common clonal origin (FIG. 51C). Mutationsclustered in CDR L1 and CDR L2 regions, and all of the frameworkregions. The most divergent sequence, gVRC-L23_(dN152), had 33 aminoacid changes, corresponding to 30.3% difference from the wild-type 10E8light chain.

Although functional, the 10E8 variants reconstituted with 10E8 wild-typecomplementary chains do not represent natural pairs. A known drawback ofthe deep sequencing approach to antibody characterization is thatseparate sequencing reactions are required for heavy and light chains,and critical information related to natural ontogeny and functionalphenotype is lost. Therefore, an evolution-based analysis was performedto provide sufficient information to recapitulate approximate naturalpairings. The maturation/evolution of heavy and light chains should belinked because of their physical association as proteins, the presenceof their evolving genes in the same cells subject to the same enzymaticmutation processes, and the requirement for cooperative structuralchange in response to the same immunogen. Furthermore, the sampling ofpaired heavy and light chains in a single cDNA library of mRNApopulation of antibody transcripts should be highly correlated becausethey originate from the same cells. In principle, this similarity insampling should lead to correlations in frequencies of correspondingheavy- and light-chain branches of phylogenetic trees.

Phylogenetic analysis of the grid-selected experimentally-tested10E8-heavy and -light chains found most of the neutralizing antibodiesto populate three branches, close to and including the template 10E8(FIGS. 50C and 50D). Branch b1-H for the heavy chain (or b1-L for thelight chain) contained 10E8, branch b2-H (b2-L) was the closest branchto b1-H, and branch b3-H (b3-L) was the next closest branch. A singleneutralizing sequence (gVRCH11_(dN152)) occupied a more distant branch(b4-H). Because 454 pyrosequencing produces on average about 5 errorsper variable antibody domain, the most potent antibody from each branchwas selected as representative, as the most functional antibody islikely to have the least 454-error-impaired function. Twelve antibodieswere reconstituted, including a complete matrix of heavy-/light-chainpairing from the 4 heavy chain branches and the 3 light chain branches(FIG. 52A). 11 out of the 12 reconstituted antibodies expressedsufficient levels of IgG to assess neutralization, which was done on apanel of five HIV-1 isolates. All 11 expressed antibodies wereneutralizing Heavy- and light-chain pairings that matched phylogeneticdistance from 10E8 (e.g. b1-H to b1-L, b2-H to b2-L, and b3-H to b3-L),were slightly more potent on average than mismatched pairings, but thedifferent was not statistically significant (FIG. 52B).

The reactivity of matched and mismatched pairing with auto-antigens wasalso tested. Notably, the matched pairing showed significantly lowerHep2 Staining (p=0.049) (FIG. 52C).

The results show with 10E8 and donor N152 (i) howidentity/divergence-grid sampling can be used to identify somaticvariants, (ii) how phylogenetic tree architecture can be used toapproximate natural pairings, and (iii) that antibodies paired byphylogenetic matching show less auto-reactivity. Such reducedauto-reactivity is likely related to in vivo selection that naturalantibodies undergo. With an antibody like 10E8, which maymechanistically be more prone to autoreactivity than other antibodies,such recapitulation of natural pairing may be useful. Although naturalpairings are lost with deep sequencing, the above suggests that it ispossible to approximate them using topological similarities betweenheavy and light chain phylogenetic trees. Thus, it may be possible touse phylogenetic similarity or lineage analysis as sieving methods, asthese provide an exclusively computational means to identify somaticvariants.

(5) Additional Variants and Combinations

Additional combinations of the identified heavy and light chains, aswell as the identified heavy and light chain variants were generated totest for neutralization and autoreactivity. Combinations of certainheavy and light chains are indicated in FIGS. 53-54, includingcombinations based on neutralization in the context of the wild-type10E8 complement chain, as well as combinations based on phylogeneticpairing. The nomenclature and sequence information for these pairings isshown in FIG. 59, except for “rL3” which corresponds to the 10E8gL03light chain germline revertant shown in FIG. 47 and listed as SEQ ID NO:149. Neutralization values (IC50 in μg/ml) against a six virus panel arepresented in FIGS. 55A-55C, and autoreactivity values are presented inFIG. 56. Neutralization values (IC50 in μg/ml) against a twenty viruspanel are presented in FIG. 57. The results indicate several heavy andlight chain pairings that show increased neutralization activitycompared to wild-type 10E8, but do not have increased autoactivity,including the pairing of heavy chain HC6 (gVRC-H2dn152; SEQ ID NO: 154)and light chain 10E8gL03 (rL3; SEQ ID NO: 152).

Further heavy chain variants were developed and complemented with 10E8light chain or 10E8 light chain variants and tested for neutralizationactivity (see FIGS. 58A and 58B). The serine residue at position 74(Kabat numbering) of the 10E8 variant heavy chain HC6 (gVRC-H2dn152; SEQID NO: 154) was additionally mutated to alanine, arginine, valine ortyrosine. The sequences of these variants are provided as SEQ ID NO: 189(HC6 S74A), SEQ ID NO: 190 (HC6 S74R), SEQ ID NO: 191 (HC6 S74V), SEQ IDNO: 192 (HC6 S74Y). These 10E8 variant heavy chains were complementedwith the “rL3” 10E8 light chain variant (10E8gL03; SEQ ID NO: 149) andthe resulting antibody was tested for neutralization activity against apanel of 20 HIV viruses, including 6, 8, 4, 1 and 1 viruses from cladesA, B, C, AG, and G, respectfully. Neutralization assays were performedas described in Example 1, and a graph summarizing the results of theseassays is presented in FIG. 58A (showing IC50 values) and FIG. 58B(showing IC80 values

Additional heavy chain and light chain mutants were developed to producegp41 binding antibodies with increased solubility, by reducing thenumber of solvent exposed hydrophobic residues. Residues that are notrequired for epitope recognition (based on 454 deep sequencing data, aswell as structure based protein redesign) were selected for substitutionwith polar or charged residues. The following 10E8-like heavy and lightchains were constructed (Kabat amino acid substitutions with referenceto the HC6 (SEQ ID NO: 154) and rL3 (SEQ ID NO: 152) heavy and lightchains, respectively)

Light Chain:

-   10E8gL03_hp_L01 (SEQ ID NO: 197; 144K)-   10E8gL03_hp_L02 (SEQ ID NO: 198; S2Y, V10T, L14A, I44V, L106P)-   10E8gL03_hp_L03 (SEQ ID NO: 199; V10T, L14A, I44K, L106P)    Heavy Chain:-   HC6_S77Y_hp_H01 (SEQ ID NO: 200; L18Q, S74Y)-   HC6_S77Y_hp_H02 (SEQ ID NO: 201; L72D, S74Y, 175K, F77T, M84T)-   HC6_S77Y_hp_H03 (SEQ ID NO: 202; L18Q, W55K, S74Y, M84T)-   HC6_S77Y_hp_H04 (SEQ ID NO: 203; L18Q, W55K, L72D, S74Y, 175K, F77T,    M84T)

A partial germline revertant construct of HC6 was also constructed:

-   HC6rH03S77Y (SEQ ID NO: 204; S23A, N73D, S74Y, E81Q, N82bS, R83K,    M84T, S87T, L89V, F91Y, R105Q)

Additional 10E8 light chain variants were constructed. These variantsinclude variants of the 10E8 wildtype light chain, the 10E8 light chainpartial germline revertant rL3, and the three light chain solubilitymutants above. These constructs contain a single mutation R23Q thatremoves a potential integrin binding site. Q is the germline identity atposition 23 in the10E8 light chain. The additional mutants are set forthas: 10E8_L_R23Q (SEQ ID NO: 205); 10E8gL03_R23Q (SEQ ID NO: 206);10E8gL03_hp_L01_R23Q (SEQ ID NO: 207); 10E8gL03_hp_L02_R23Q (SEQ ID NO:208); 10E8gL03_hp_L03_R23Q (SEQ ID NO: 209). The person of ordinaryskill in the art will appreciate that the 10E8 or any of the 10E8variant light chain variable regions disclosed herein can optionallyinclude the R23Q substitution.

(6) Summary of 10E8 Mutations.

The 10E8 substitutions described above are summarized in the tablesgiven as FIGS. 60A and 60B (heavy chain substitutions) and FIGS. 61A and61B (light chain substitutions).

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described embodiments. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

We claim:
 1. An isolated human monoclonal antibody, comprising: a heavychain variable region comprising a heavy chain complementaritydetermining region (HCDR)1, a HCDR2, and a HCDR3 comprising amino acids26-33, 51-60, and 99-120 of SEQ ID NO: 1, respectively; and a lightchain variable region comprising a light chain complementaritydetermining region (LCDR)1, a LCDR2, and a LCDR3, comprising amino acids26-31, 49-51, and 88-99 of SEQ ID NO: 2, respectively; wherein theantibody specifically binds gp41 and neutralizes HIV-1 infection.
 2. Theisolated human monoclonal antibody of claim 1, wherein the heavy chainvariable region comprises the amino acid sequence set forth as one ofSEQ ID NOs: 1, 3, 5, 149, 154, 189-192, 200-201, or 204, and furthercomprises at most ten amino acid substitutions in framework regions ofthe heavy chain variable region.
 3. The isolated human monoclonalantibody of claim 1, wherein the heavy chain variable region comprisesthe amino acid sequence set forth as SEQ ID NO: 11, wherein X₁ is Q orR, X₂ is V or A, X₃ is S or Y, and X₄ is T or I.
 4. The isolated humanmonoclonal antibody of claim 1, wherein the heavy chain variable regionof the antibody comprises the amino acid sequence set forth as one ofSEQ ID NOs: 1, 3, 5, 149, 154, 189-192, 200-201, or
 204. 5. The isolatedhuman monoclonal antibody of claim 1, wherein the light chain variableregion comprises the amino acid sequence set forth as one of SEQ ID NOs:2, 4, 150-152, or 164-168.
 6. The isolated human monoclonal antibody ofclaim 1, wherein the light chain variable region comprises the aminoacid sequence set forth as one of SEQ ID NOs: 2, 4, 150-152, or 164-168,and further comprises at most ten amino acid substitutions in frameworkregions of the light chain variable region.
 7. The isolated humanmonoclonal antibody of claim 1, wherein: the heavy chain variable regioncomprises the amino acid sequence set forth as SEQ ID NO: 1, and thelight chain variable region comprises the amino acid sequence set forthas SEQ ID NO: 2; the heavy chain variable region comprises the aminoacid sequence set forth as SEQ ID NO: 154, and the light chain variableregion comprises the amino acid sequence set forth as SEQ ID NO: 152; orthe heavy chain variable region comprises the amino acid sequence setforth as SEQ ID NO: 192, and the light chain variable region comprisesthe amino acid sequence set forth as SEQ ID NO:
 152. 8. The isolatedhuman monoclonal antibody of claim 1, wherein the antibody is an IgG,IgM or IgA antibody.
 9. The isolated human monoclonal antibody of claim1, wherein the antibody neutralizes at least 98% of the HIV-1 isolateslisted in FIGS. 17C-17F with an inhibitory concentration (IC50) of lessthan 50μg/ml.
 10. The isolated human monoclonal antibody of claim 1,wherein the antibody neutralizes at least 72% of the HIV-1 isolateslisted in FIGS. 17C-17F with an inhibitory concentration (IC50) of lessthan 1μg/ml.
 11. A bispecific antibody comprising the isolated humanmonoclonal antibody of claim
 1. 12. An antigen binding fragment of theisolated human monoclonal antibody of claim
 1. 13. The antigen bindingfragment of claim 12, wherein the fragment is a Fab fragment, a Fab’fragment, a F(ab)'₂ fragment, a single chain Fv protein (scFv), or adisulfide stabilized Fv protein (dsFv).
 14. The antigen binding fragmentof claim 13, wherein the fragment is a Fab fragment.
 15. The isolatedantigen binding fragment of claim 12, linked to an Fc domain and/orIL-15.
 16. The isolated human monoclonal antibody of claim 1, linked toan effector moiety.
 17. The isolated human monoclonal antibody claim 16,wherein the effector moiety is a toxin or a detectable label.
 18. Acomposition comprising: (a) the antibody of claim 1; and (b) apharmaceutically acceptable carrier.
 19. A kit comprising: (a) theantibody of claim 1; and (b) instructions for using the kit.
 20. Theisolated human monoclonal antibody of claim 1, wherein the heavy chainvariable region comprises the amino acid sequence set forth as SEQ IDNO:
 1. 21. The isolated human monoclonal antibody of claim 1, whereinthe light chain variable region comprises the amino acid sequence setforth as SEQ ID NO:
 2. 22. The isolated human monoclonal antibody ofclaim 1, wherein the heavy chain variable region comprises the aminoacid sequence set forth as SEQ ID NO: 1, and the light chain variableregion comprises the amino acid sequence set forth as SEQ ID NO: 2.